Crosslinkable polysaccharide derivative, process for producing the same, crosslinkable polysaccharide composition, and medical treatment material

ABSTRACT

A crosslinkable polysaccharide derivative which has in the side chain of polysaccharide at least one active ester group reactive with an active hydrogen-containing group and which forms a crosslinked product through covalent bond between the active ester group and the active hydrogen-containing group upon contact with alkaline water. A composition and a medical treatment material containing the crosslinkable polysaccharide derivative. The crosslinkable polysaccharide derivative produces a high bond strength that meets clinical requirements. It avoids the risk of infection because it is based on a material which is not derived from living organisms. It in itself and its decomposition products have a low level of toxicity because it is formed from a synthetic material. It is biodegradable and bioabsorbable. It can be prepared readily and simply without requiring special apparatus at the time of use.

This disclosure is based upon Japanese Application No. 2003-044861,filed Feb. 21, 2003, Japanese Application No. 2003-111504, filed Apr.16, 2003, Japanese Application No. 2004-001184, filed Jan. 6, 2004, andInternational Application No. PCT/JP2004/001958, filed Feb. 20, 2004,the contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a crosslinkable polysaccharidederivative to be used under a specific condition, a process forproducing the same, a composition containing the polysaccharidederivative, and a medical treatment material based on the composition.More particularly, the present invention relates to a use of acrosslinkable polysaccharide derivative under alkaline conditions whichhas an active ester group introduced thereinto, and thereby which iscapable of self-crosslinking through binding with an intramolecularhydroxy group and is also capable of adhesion to the surface of anorganism through binding with an active hydrogen-containing group on thesurface of an organism. The present invention relates also to a processfor producing a polysaccharide derivative which is essentially withbiosafety and chemical safety because of absence of any organism-derivedmaterial or any potentially toxic chemical substance, therefore which isa highly biocompatible material. Moreover, the present invention relatesalso to a crosslinkable polysaccharide composition and a medicaltreatment material both containing the polysaccharide derivative.Particularly, the medical treatment material is easy to prepare at thetime of use, exhibits good adhesion properties to the surface of anorganism (as adherend), and forms a flexible crosslinked product whichfollows easily with a compiant the deformation of the adherend. Hence itis suitable for use as a hemostatic material and a medical adhesive.

BACKGROUND ART

Medical treatment materials play an important role in surgery, reducingtime for operation. An example of medical treatment materials is anadhesive for living tissue, which is used to adhere or block vessels ororgan tissues to stop leakage of body fluid such as blood, lymph, or gasfrom sutured or coated tissue.

Among clinically successful and popular adhesives for living tissue isfibrin glue. (See, for example, “Bonding of Living Tissue” by ShojiroMatsuda, “Adhesion” (Japan), vol. 44, No. 1, pp. 19 to 27, Jan. 25,2000, issued by Kobunshi Kankokai.). Fibrin glue is a two-packhemostatic material that utilizes the principle of blood coagulation. Itworks as follows. Fibrinogen is converted into fibrin through theenzymatic action of thrombin. Then, Factor VIII which has been activatedby thrombin crosslinks fibrin, thereby forming a fibrin clump. Thedisadvantage of fibrin glue is the possibility of virus infectionthrough its use, because fibrinogen, Factor VIII, and thrombinconstituting fibrin glue are materials derived from living organisms(among organism materials, material from organism such as human andanimal) and the quality control for initial material and safetyprecaution such as inactivation or removal of virus during manufactureare not always complete. Moreover, fibrin glue is expensive, weak inadhesive strength, and complex to handle. Therefore, attempts have beenmade to find its substitute.

The above-mentioned thesis “Bonding of Living Tissue” also mentions atechnique using a gelatin glue (GRF adhesive) as another adhesive forliving tissue which has been clinically used with success. GRF is amixture of gelatin and resorcinol which is to be crosslinked withformaldehyde and glutaraldehyde. It is characterized by high adhesivestrength. It is used in the same way as fibrin glue, and it is also usedfor filling and adhering of a sac resulting from dissecting aneurysm ofthe aorta. However, it is said to induce a prevention of restorationtissue in the neighborhood of application part because formaldehyde istoxic in itself.

In view of the problems of the biological safety such as infection, inrecent years, synthetic material-based tissue adhesives using nomaterial derived from living organisms have been actively developed andsome adhesives have been proposed. An example is an ethyl or isobutylcyanoacrylate adhesive among 2-cyanoacrylate adhesives widely used forthe instant adhesive (“Surgery of Stanford Type A Dissecting Aneurysm ofAorta” S. Kawada et al., “Surgical Practice” (Japan), Shindan toChiryou-sha, vol. 32, No. 9, pp. 1250 to 1258, Sep. 1, 1990). Thisadhesive is characterized by its rapid adhesion and high adhesionstrength because it rapidly polymerizes and cures for adhesion usingmoisture as a polymerization initiator. However, its cured product isharder than the corresponding living tissue and has no excellent abilityto follow the movements of the living tissue. The toxicity problem offormaldehyde generated by the hydrolysis of the cured product in theliving organism has also been raised.

One way to obviate these problems is proposed in JP 62-290465 A whichdescribes an adhesive for surgical treatment that includes as a maincomponent an NCO-terminated hydrophilic urethane prepolymer composed ofp-phenylene diisocyanate (PPDI) and hydrophilic polyether polyol. In theurethane-based adhesive, first, the isocyanate group at both terminalsreact with water to generate carbon dioxide gas and to be converted toan amino group. Then, part of the produced amine group react with theisocyanate group and an amino group in living tissue protein also reactwith the isocyanate group to form urethane linkages, whereby theadhesive is cured to adhere to the living tissue. Since the curedproduct of the adhesive is flexible, the adhesive is capable offollowing the movements of living organism. However, the cured productis hardly biodegradable, may cause infection because it remains for along time and thus has the problem of biodegradability andbioabsorbability.

On the other hand, synthetic tissue adhesives having high adhesionstrength and excellent biodegradability and bioabsorbability have alsobeen developed and applied to clinical treatments. An example is aphotopolymerizable bioabsorbable adhesive (trade name: Advaseal™), whichis mentioned in “Experience in Using Photopolymerizable AbsorbableHydrogel (Advaseal™)—Clinical Application”, M. Takagi et al., “ThoracicSurgery” (Japan), Nankodo, vol. 53, No. 11, pp. 951 to 953, Oct. 1,2000. In this adhesive, acryl ester terminal group is bound to acopolymer of polyethylene glycol (primer) and polylactic acid or acopolymer of polyethylene glycol (primer) and trimethylene carbonate(sealant) and the adhesive also includes an eosin dye as aphotosensitive substance. The primer is applied to a wound. Then, thesealant is applied to the wound to which the primer has been applied.Thereafter, the wound is irradiated with light (450 to 550 nm inwavelength) from a xenon light source for about 40 seconds. Irradiationbrings about photopolymerization, which causes the preparation to bepolymerized and cured into a hydrogel, whereby the preparation adheresto the living tissue. The hydrogel is gradually absorbed into the livingbody and eventually it disappears about nine months after theapplication. However, it is necessary to prepare an apparatus for lightirradiation on an operating table to use the adhesive, which has a spacelimitation. The economic burden for the apparatus installation andmaintenance is also heavy.

U.S. Pat. No. 6,323,278, JP 2000-502380 and JP 2002-541923 propose othermethods in which a two-component mixture type crosslinking materialcomposed of synthetic polymers each having different groups reactingwith each other is applied to a tissue to form a crosslinked polymermatrix. More specifically, a first component having a nucleophilic groupsuch as a primary amino group or a thiol group introduced in themolecular chain terminals of polyethylene glycol of a multi-branchedstructure is mixed with a second component having an electrophilic groupsuch as a succinimidyl group introduced therein to form a crosslinkedgel (hydrogel) (U.S. Pat. No. 6,323,278). The polyethylene glycol as theskeleton of each component is one which has a weight-average molecularweight of 10,000, so that the crosslinked product decomposes into smallmolecules that can be eliminated through kidneys. The adhesive isdesigned with biodegradation and bioabsorption taken into consideration.However, the two components have to be prepared separately in the formof solution, sprayed through separate ports of an applicator to be mixedtogether before use and applied to the wound. Therefore, they have to beprepared previously in anticipation of the timing of use duringoperation. It is difficult to immediately cope with the application.

In the meantime, it is known that polysaccharides are highlybiocompatible materials. In particular, U.S. Pat. No. 5,676,964 and WO00/27886 propose crosslinked products of polysaccharides such ashyaluronic acid having a carboxy group in the molecule. The crosslinkedproducts of polysaccharides are formed by using an intramolecularcarboxy group activated by carbodiimide, ethoxyacetylene, Woodwardreagent, chloroacetonitrile (U.S. Pat. No. 5,676,964), and an activatorused in peptide chemistry (WO 00/27886). For the method of crosslinkingactivated polysaccharides, are disclosed a crosslinking method byheating or UV light irradiation (U.S. Pat. No. 5,676,964) and acrosslinking method using polyamine (WO 00/27886).

The above-mentioned publications propose the use of the crosslinkedproducts in the form of film, sponge, capsule, tablet, and DDS carrierfor medicine and surgery, but do not disclose the use of the activatedpolysaccharides in uncrosslinked form.

In polysaccharide activation, a carboxy group in the polysaccharide isin the form of salt such as an ammonium salt prior to the reaction withan activator. Inactivated carboxyl residues after the polysaccharideactivation are in the form of sodium salt or the like. Therefore, thereis a high possibility that the crosslinked product contains residualammonium or metallic salt.

There is a related art technique (WO 95/24429) which discloses a methodof activating a polysaccharide having carboxylic acid in the molecule inthe form of salt as mentioned above. It discloses an activatedpolysaccharide which is formed from a polysaccharide such as hyaluronicacid having a carboxy group in the molecule, by partial or completeesterification with an aromatic alcohol, heteroaromatic alcohol, orN-hydroxylamine alcohol. The activated polysaccharide will find use asan intermediate for peptide synthesis. However, the use of the activatedpolysaccharide in its uncrosslinked state is not disclosed, as in theabove-mentioned patent documents.

As mentioned above, the medical treatment materials typified by tissueadhesives which are used in a living body should meet clinicalrequirements for not only adhesion strength but also safety, and it isimportant to design the materials taking into account avoidance ofinfection by the use of materials not derived from living organism,reduction of the toxicity of the components or a decomposed product bythe use of a synthetic material, and biodegradability andbioabsorbability. Moreover, they should be available at any time whennecessary without requiring preliminary steps during operation andwithout requiring special equipment for their use.

DISCLOSURE OF INVENTION

The present invention was completed to realize the medical treatmentmaterial that meets the above-mentioned requirements, and this object isachieved by a polysaccharide derivative in a new using manner. It is anobject of the present invention to provide a crosslinkable materialcontaining the polysaccharide derivative, a process for producing thesame, and a medical treatment material that can be used whenevernecessary with simple preliminary steps without requiring specialequipment.

The present invention covers the following items (1) to (48).

(1) A crosslinkable polysaccharide derivative for forming a crosslinkedproduct, wherein said crosslinkable polysaccharide derivative has atleast one active ester group which has been introduced into the sidechain of said polysaccharide and which is reactive with an activehydrogen-containing group, and said crosslinked product has a covalentbond between said active ester group and the active hydrogen-containinggroup upon contact with water under alkaline conditions.

(2) The crosslinkable polysaccharide derivative according to (1) above,wherein the active hydrogen-containing group is an intramolecularhydroxy group of polysaccharide and said polysaccharide derivative isselfcrosslinkable.

(3) The crosslinkable polysaccharide derivative according to (1) or (2)above, wherein the active hydrogen-containing group is that on thesurface of a living organism and the polysaccharide is capable ofadhesion to the surface of a living organism.

(4) The crosslinkable polysaccharide derivative according to any one of(1) to (3) above, wherein the active ester group is one in which anelectrophilic group attached to a carbonyl group therein.

(5) The crosslinkable polysaccharide derivative according to (4) above,wherein the electrophilic group is one which is introduced from anN-hydroxylamine compound.

(6) A polysaccharide (A) according to any one of (1) to (5) above,wherein the active ester group is a succinimide ester group.

(7) The crosslinkable polysaccharide derivative according to any one of(1) to (6) above, wherein the polysaccharide derivative contains theactive ester group in an amount of 0.1 to 2 mmol/g based on dry weight.

(8) The crosslinkable polysaccharide derivative according to any one of(1) to (7) above, wherein the polysaccharide derivative further containsa carboxy group and/or a carboxyalkyl group.

(9) The crosslinkable polysaccharide derivative according to any one of(1) to (8) above wherein the crosslinkable polysaccharide derivative isof non-salt type.

(10) The crosslinkable polysaccharide derivative according to any one of(1) to (9) above, wherein a starting polysaccharide to which to beintroduced said active ester group and which has a carboxy group and/ora carboxyalkyl group, as a precursor state of said crosslinkablepolysaccharide derivative, is of non-salt form and is a polysaccharidedissoluble in an aprotic polar solvent at a temperature ranging from 60°C. to 120° C.

(11) The crosslinkable polysaccharide derivative according to any one of(1) to (10) above, wherein said starting polysaccharide to be introducedsaid active ester group thereto is a polysaccharide which does not own acarboxy group and a carboxyalkyl group.

(12) The polysaccharide derivative according to (11) above, wherein thestarting polysaccharide is at least one selected from the groupconsisting of dextran and pullulan.

(13) The polysaccharide derivative according to any one of (1) to (12)above, wherein the starting polysaccharide into which the active estergroup is to be introduced is pectin and/or hyaluronic acid. Thisstarting polysaccharide per se is an active esterified precursor(polysaccharide containing an acid group).

(14) The crosslinkable polysaccharide derivative according to any one of(1) to (13) above, wherein pH of the alkaline conditions is ranging from7.5 to 12.

(15) The crosslinkable polysaccharide derivative according to any one of(1) to (14) above which is in the form of powder.

(16) The crosslinkable polysaccharide derivative according to any one of(1) to (14) above which is in the form of uncrosslinked sheet.

(17) The crosslinkable polysaccharide derivative according to (16)above, wherein the sheet is a heat-dried film or a freeze-dried sheet.

(18) A process for producing a crosslinkable polysaccharide derivativehaving an active ester group

12. A process for producing a crosslinkable polysaccharide derivativehaving an active ester group, said process comprises:

dissolving an acid-containing polysaccharides (a precursor of acrosslinkable polysaccharide derivative) having a carboxy group and/or acarboxyalkyl group which are originally possessed or which have beenintroduced, in its non-salt form, into an aprotic polar solvent at atemperature ranging from 60° C. to 120° C., and

reacting it with an electrophilic group-introducing reagent in thepresence of a dehydrating-condensing agent, and thereby converting atleast part of said carboxy group and/or carboxyalkyl group into activeesters.

(19) The process for producing a crosslinkable polysaccharide derivativeaccording to (18) above, wherein the acid-containing polysaccharidecontains the carboxy group and/or the carboxyalkyl group in an amount of0.1 to 5 mmol/g based on dry weight.

(20) The process according to (18) or (19) above, wherein thedehydrating-condensing agent is used in an amount (Z mmol) defined by0.1<(Z/X)<50, where X denotes the amount (X mmol) of the carboxy groupand/or the carboxyalkyl group in the acid-containing polysaccharidepresenting in reaction system.

(21) The process according to any one of (18) to (20) above, whichfurther comprises a step of purifying and/or drying the previouslyobtained polysaccharide derivative.

(22) The process according to any one of (18) to (21) above, wherein theaprotic polar solvent is dimethylsulfoxide.

(23) The process according to any one of (18) to (22) above, wherein theelectrophilic group-introducing agent is an N-hydroxylamine compound.

(24) The process according to (23) above, wherein the N-hydroxylaminecompound is N-hydroxysuccinimide.

(25) The process according to any one of (18) to (24) above, wherein thedehydrating-condensing agent is 1-ethyl-3-dimethyaminopropylcarbodiimidehydrochloride.

(26) The process according to any one of (18) to (25) above, whichfurther comprises steps of preparing an aqueous solution of thepreviously obtained polysaccharide derivative and developing to form itinto an uncrosslinked sheet with desired shape by heat-drying orfreeze-drying.

(27) A crosslinkable polysaccharide composition which comprises thecrosslinkable polysaccharide derivative (A) according to any one of (1)to (17) above and a polymer (C) other than the polysaccharide derivative(A).

(28) The crosslinkable polysaccharide composition according to (27)above, wherein the polymer (C) is a polymer which has two or moreprimary amino groups and/or thiol groups in one molecule.

(29) The crosslinkable polysaccharide composition according to (27) or(28) above, wherein the polymer (C) is at least one selected frompolyalkylene glycol derivatives, polypeptides, and polysaccharides andderivatives thereof.

(30) The crosslinkable polysaccharide composition according to (29)above, wherein the polyalkylene glycol derivative is at least oneselected from the group consisting of polyethylene glycol (PEG)derivative, polypropylene glycol derivative, polybutyrene glycolderivative, and polypropylene glycol-polyethylene glycol block copolymerand random copolymer derivatives.

(31) The crosslinkable polysaccharide composition according to (30)above, wherein the main polymer backbone of the polyethylene glycolderivative is at least one selected from the group consisting ofethylene glycol, trimethylol ethane, diglycerol, pentaerythritol, andhexaglycerol, and has a molecular weight of 100 to 50,000.

(32) The crosslinkable polysaccharide composition according to (30)above, wherein the polyethylene glycol derivative is one which is atleast one selected from the group consisting of ethylene glycol-typepolyethylene glycol derivative having a thiol group on both terminalsand having a weight-average molecular weight of 1,000, 2,000, 6,000, or10,000, ethylene glycol-type polyethylene glycol derivative having anamino group on both terminals and having a weight-average molecularweight of 1,000, 2,000, 6,000, or 10,000, trimethylol ethane-typepolyethylene glycol derivative having a thiol group on three terminalsand having a weight-average molecular weight of 5,000 or 10,000,trimethylol ethane-type polyethylene glycol derivative having an aminogroup on three terminals and having a weight-average molecular weight of5,000 or 10,000, diglycerol-type polyethylene glycol derivative having athiol group on four terminals and having a weight-average molecularweight of 5,000, 10,000, or 20,000, diglycerol-type polyethylene glycolderivative having an amino group on four terminals and having aweight-average molecular weight of 5,000, 10,000, or 20,000,pentaerythritol-type polyethylene glycol derivative having a thiol groupon four terminals and having a weight-average molecular weight of 10,000or 20,000, pentaerythritol-type polyethylene glycol derivative having anamino group on four terminals and having a weight-average molecularweight of 10,000 or 20,000, hexaglycerol-type polyethylene glycolderivative having a thiol group on eight terminals and having aweight-average molecular weight of 10,000 or 20,000, andhexaglycerol-type polyethylene glycol derivative having an amino groupon eight terminals and having a weight-average molecular weight of10,000 or 20,000.

(33) The crosslinkable polysaccharide composition according to (29),wherein the polypeptide is at least one selected from the groupconsisting of collagen, gelatin, albumin, and polylysine.

(34) The crosslinkable polysaccharide composition according to (29),wherein the polysaccharide is at least one selected from the groupconsisting of pectin, hyaluronic acid, chitin, chitosan,carboxymethylchitin, carboxymethylchitosan, chondroitin sulfate, keratinsulfate, kerato sulfate, heparin, and derivatives thereof.

(35) The crosslinkable polysaccharide composition according to any oneof (27) to (34) above, which contains the crosslinkable polysaccharidederivative (A) and the polymer (C) separated in the form of aqueoussolution.

(36) The crosslinkable polysaccharide composition according to any oneof (27) to (34) above, which is in the form of powder.

(37) The crosslinkable polysaccharide composition according to any oneof (27) to (34) above, which is in the form of uncrosslinked sheet.

(38) A sheet-like product according to (37) above, which is a compositesheet comprising a sheet of polysaccharide derivative according to (16)or (17) above and a sheet of the polymer (C) attached thereto.

(39) A method for producing the sheet-like product according to (37) or(38) above, which comprises attaching a sheet of the polymer (C) to anuncrosslinked sheet of the polysaccharide derivative (A) obtained in(26) above.

(40) The method for producing the sheet-like product according to (39)above, wherein the polymer (C) is dissolved in a nonaqueous volatileorganic solvent and the resulting solution is impregnated into theuncrosslinked sheet-like product of the polysaccharide derivative (A)and dried.

(41) A crosslinkable polysaccharide composition which includes thepolysaccharide derivative (A) according to any one of (1) to (17) aboveand a pH adjusting agent (B) which is not mixed with the polysaccharidederivative (A).

(42) The crosslinkable polysaccharide composition according to any oneof (27) to (35) above, which further includes a pH adjusting agent (B)which is not mixed with the polysaccharide derivative (A).

(43) The crosslinkable polysaccharide composition according to (35)above, wherein the aqueous solution of the polymer (C) contains the pHadjusting agent (B).

(44) A medical treatment material which comprises either thecrosslinkable polysaccharide derivative according to any one of (1) to(17) above or the crosslinkable polysaccharide composition according toany one of (27) to (43) above.

(45) The medical treatment material according to (44) above, which is ahemostatic material and/or a biomedical adhesive.

(46) The medical treatment material according to (44) or (45) above,which is in the form of aerosol or paste.

(47) A kit which includes the medical treatment material according toany one of (44) to (46) above.

(48) A method for restraining hemostasis and/or adhering of livingorganism, which comprises reacting either the crosslinkablepolysaccharide derivative according to any one of (1) to (17) above orthe crosslinkable polysaccharide composition according to any one of(27) to (43) above at a desired part in the presence of water underalkaline conditions.

The polysaccharide derivative according to the present invention isessentially with biosafety and chemical safety and hence highlybiocompatible in itself because it is not based on any organism-derivedmaterial or potentially toxic chemical substance. Moreover, since thepolysaccharide derivative is self-crosslinkable and capable of adhesionto the surface of a living organism under alkaline conditions, and canbe easily prepared at the time of use without requiring any specialequipment, and may sufficiently adhere to the surface of a livingorganism and forms a crosslinked product which is flexible enough tofollow the deformation of adherent, it is suitable for use as a medicaltreatment material such as a hemostatic material or a biomedicaladhesive.

BEST MODE FOR CARRYING OUT THE INVENTION

A detailed description is given below of the crosslinkablepolysaccharide derivative, the crosslinkable polysaccharide compositionand the medical treatment material containing it, and the process forproducing them according to the present invention.

The crosslinkable polysaccharide derivative of the present invention hasat least one active ester group, which has been introduced into the sidechain of the polysaccharide, and which is reactive with an activehydrogen-containing group. The polysaccharide (the starting material) towhich to be introduced the active ester group will be described later,the polysaccharide molecule originally has an hydroxy group i.e. anactive hydrogen-containing group, therefore, the polysaccharide havingthe active ester group introduced thereto has both the active estergroup and the active hydrogen-containing group in one molecular chainand is self-crosslinkable under the reaction conditions. Theself-crosslinking means an intramolecular or intermolecular reactionbetween an active ester group and an active hydrogen-containing group ofthe polysaccharide derivative to form the covalent bond. In a case ofutilizing an active hydrogen-containing group on the surface of livingorganism, the crosslinkable polysaccharide derivative may adhere to thesurface of living organism.

In this specification, such crosslinkable polysaccharide derivativementioned above may be referred to as active esterified polysaccharideor simply polysaccharide derivative herein after.

Incidentally, the term molecule used in “one molecular chain” or“intramolecular” means one molecule consisting of one region connectedwith serial bonds by covalent bond.

The polysaccharide derivative of the present invention is an activeesterified polysaccharide, and essentially retains the skeleton ofpolysaccharide. Therefore, the polysaccharide derivative will bedescribed below together with the active-esterifying method (the processfor producing the polysaccharide derivative).

In the present invention, the active ester group to be introduced intothe polysaccharide is not specifically restricted so long as it formscovalent bond upon reaction with an active hydrogen-containing group inthe presence of water under alkaline conditions. This active ester groupis a group in which an electrophilic group stronger than an ordinaryester bound to the carbonyl carbon of a carboxy group or a methylcarboxygroup which is ordinarily owned by the polysaccharide or introduced uponconversion into acid type. To be concrete, the active ester group may berepresented by —COOX, —OX representing the alcohol moiety is from theelectrophilic group which is preferably a group introduced from anN-hydroxylamine compound. The N-hydroxylamine compound is acomparatively inexpensive raw material and hence it is easy in operationfor introduction of the active ester group on an industrial scale.

The N-hydroxylamine compound to form the —OX moiety includes, forexample, N-hydroxysuccinic imide, N-hydroxynorbornene-2,3-dicarboxylicimide, ethyl ester of 2-hydroxyimino-2-cyanoacetate,2-hydroxyimino-2-cyanoacetic amide, and N-hydroxypiperidine.

In the present invention, the active ester group in the polysaccharidederivative may have one or more species.

Of these active ester groups, the succinimide ester group is desirable.

The polysaccharide derivative of the present invention has at least oneactive ester group mentioned above in the molecule, however, generally,it has two or more active ester groups in the molecule so that it formsthe crosslinked matrix. The amount of the active ester group per onegram of polysaccharide derivative based on dry weight should preferablybe 0.1 to 2 mmol/g, which varies depending on the purpose of use.

In the present invention, the polysaccharide constituting the mainskeleton of the polysaccharide derivative, having the active ester groupintroduced thereinto, is not specifically restricted so long as it hastwo or more units of monosaccharide structure in the main skeleton. Suchpolysaccharides include those formed through covalent bond frommonosaccharides such as arabinose, ribose, xylose, glucose, mannose,galactose, fructose, sorbose, rhamnose, furcose, and ribodesose;disaccharides such as trehalose, sucrose, maltose, cellobiose,gentiobiose, lactose, melibiose; and tri or more polysaccharides such asraffinose, gentianose, merezitose, and stachyose; those having furtherfunctional groups introduced thereinto. In the present invention, suchpolysaccharide may be naturally occuring ones or artificiallysynthesized ones. Also, the polysaccharide derivative of the presentinvention may have the skeleton of one polysaccharide or two or morepolysaccharides.

The polysaccharide constituting the main skeleton of the polysaccharidederivative of the present invention is not specifically restricted inweight-average molecular weight. It should preferably have aweight-average molecular weight of 5,000 to 2,500,000, which correspondsto that of a polysaccharide composed of tens to thousands ofmonosaccharides, disaccharides, or tri or more, and polysaccharidesmentioned above. Such a polysaccharide gives rise to a gel (whichresults from the polysaccharide derivative of the present inventionthrough crosslinking) that permits easy adjustment of hardness, becauseit also permits easy introduction of more than one active ester groupand active hydrogen-containing group into one molecular chain. Adesirable polysaccharide is one which has a weight-average molecularweigh of 10,000 to 1,000,000.

The starting polysaccharide constituting the main skeleton of thepolysaccharide derivative should preferably be a polysaccharide havingthe above constituent, which has a carboxylic acid group to form theactive ester group“—COOX” in the precursor state prior to activeesterification (It may be referred to as an acid group-containingpolysaccharide hereinafter.). The carboxylic acid group denotes acarboxy group and/or carboxyalkyl group (which may be collectivelyreferred to as a carboxylic acid group hereinafter). The carboxyalkylgroup is a functional group in which carboxy group binds to the alkylskeleton, as which includes carboxymethyl group, carboxyethyl group,carboxypropyl group, carboxyisopropyl group, and carboxybutyl group.

The above-mentioned starting polysaccharide is not specificallyrestricted so long as it is an acid group-containing polysaccharide inthe precursor state of the crosslinkable polysaccharide derivative. Itmay be a native polysaccharide having a carboxylic acid. It may also bea polysaccharide which does not originally have a carboxylic acid buthas a carboxy group and/or carboxyalkyl group introduced thereinto. Ofthese carboxylic acid group-containing polysaccharides, the followingare desirable. Natural polysaccharide having a carboxy group.Carboxylated polysaccharide having a carboxy group introduced thereinto.Carboxymethylated polysaccharide having a carboxymethyl group introducedthereinto. Carboxyethylated polysaccharide having a carboxyethyl groupintroduced thereinto. The following are particularly desirable. Naturalpolysaccharide having a carboxy group, carboxylated polysaccharidehaving a carboxy group introduced therein, and carboxymethylatedpolysaccharide having a carboxymethyl group introduced thereinto.

The above-mentioned native polysaccharide having a carboxylic acid isnot specifically restricted. It includes pectin (which containsgalacturonic-acid) and hyaluronic acid. Pectin is commercially availableunder a trade name of “GENUE Pectin” from CP Kelco (Denmark) andhyaluronic acid is commercially available under a trade name of“Hyaluronic acid FCH” from Kibun Food Inc. (Japan). Pectin is apolysaccharide composed mainly of galacturonic acid (about 75 to 80%),the remainder being other sugars. In other words, pectin is apolysaccharide composed of galacturonic acid and other sugars in theabove-mentioned ratio. Hyaluronic acid is used for ophthalmic surgeryadjuvant and therapy eutic agent of degenerative osteoarthrosis.Hyaluronic acid does not contain galacturonic acid.

In the present invention, the carboxy group and/or carboxyalkyl group inthe polysaccharide derivative is preferably “non-salt” type; in otherwords non-coordinated salt, and, it is desirable that the finallyresulting polysaccharide derivative is not in the form of salt. The term“salt” includes inorganic salt of alkali metal or alkaline earth metal,quaternary amine salt such as tetrabutyl ammonium (TBA), and halogenatesalt such as chloromethyl pyridilium iodide. The term “non-salt” typemeans that the derivative does not have such a “salt”. The term “not inthe form of salt” means that the derivative does not contain such asalt.

The polysaccharide into which to be introduced the above-mentionedcarboxy group and/or carboxyalkyl group are, but is not specificallyrestricted, may include dextran and pullulan.

Dextran is used as a blood plasma substitute. Dextran may include“Dextran T fractions” from Amersham Biosciences (Japan), and Pullulanmay include “Pullulan PI-20” from Hayashibarashasha (Japan). Pullulan isused as a medical adjuvant including an oral medication. Preferable oneis free of biological contamination such as endotoxin.

In the present invention, any one which is commercially available ingeneral may be used for each polysaccharide. Those polysaccharides whichhave been used for therapy as mentioned above are suitable for use inthe present invention from the view point of safety.

The carboxylation reaction of polysaccharide may be accomplished by anyknown oxidation reaction without specific restrictions. Type ofcarboxylation reaction, but is not specifically restricted, includesoxidation with dinitrogen tetraoxide, oxidation with fusing sulfuricacid, oxidation with phosphoric acid, oxidation with nitric acid, andoxidation with hydrogen peroxide. Each oxidation with the reagent willbe accomplished by selecting the reaction known in ordinary. Thecondition of reaction may be properly established depending on theamount of carboxy group to be introduced. For example, a carboxylatedpolysaccharide (a carboxylated form of polysaccharide) may be preparedthrough the oxidation of the hydroxy group in a polysaccharide bysuspending a polysaccharide as a starting material into chloroform orcarbon tetrachloride and adding a dinitrogen tetraoxide thereto.

Additionally, a carboxylalkylation reaction may be accomplished by anyknown carboxyalkylation reaction of polysaccharide, but is notspecifically restricted, typically in the case of carboxymethylation,may be applied of the reaction which uses monochloroacetic acid afteralkalifying of polysaccharide. The condition of reaction may be properlyestablished depending on the amount of carboxymethyl group to beintroduced.

In the present invention, it is possible to employ either thecarboxylation or the carboxyalkylation mentioned above as a method forintroducing the carboxylic acid group into a polysaccharide, but is notlimited, carboxyalkylation, especially carboxymethylation is suitablebecause it does not appreciably reduce the molecular weight ofpolysaccharide after introduction of the carboxy group and it permitseasy control over the amount of the carboxy group to be introduced.

In the present invention, introduction of the carboxylic acid group isnot restricted to polysaccharide which does not originally have thecarboxylic acid group. Introduction of carboxy group and/orcarboxymethyl group may be performed on native polysaccharide, such ashyaluronic acid mentioned above, which originally has the carboxylicacid group.

One or more than one acid group-containing polysaccharide may be used toactive-esterify the carboxy group and/or carboxymethyl group therein.

The acid group-containing polysaccharide to be active-esterified shouldbe one which contains carboxylic acid group (regarded as one molecule)in an amount of usually 0.1 to 5 mmol/g, preferably 0.4 to 3 mmol/g,more preferably 0.6 to 2 mmol/g (on dry basis). If the amount ofcarboxylic acid group is less than 0.1 mmol/g, it is often the case thatthere will be an insufficient number of active ester group for forming acrosslinking point derivated therefrom. If the amount of carboxylic acidgroup is more than 5 mmol/g, the polysaccharide (uncrosslinked) ispoorly soluble in a water-containing solvent.

The method of active-esterifying the acid group-containingpolysaccharide (the method for producing the polysaccharide derivative)is not specifically restricted. It may be the one which involvesreacting the acid group-containing polysaccharide with an electrophilicgroup-introducing agent in the presence of a dehydrating-condensingagent, or it may be the one which involves ester exchange reaction tointroduce an active ester group into the polysaccharide from a compoundhaving an active ester group. The former method is suitable for thepresent invention, and this method will be described in the following.(It will be referred to as the method of the present invention.)

The method of the present invention is accomplished usually bydissolving the acid group-containing polysaccharide in an aprotic polarsolvent and using the resulting solution for reaction. To be concrete,the method comprises a step of dissolving a polysaccharide having acarboxy group or a carboxyalkyl group in an aprotic polar solution,thereby preparing a solution, a step of adding to the solution anelectrophilic group-introducing agent and a dehydrating-condensingagent, thereby active-esterifying the carboxy group or carboxyalkylgroup of the polysaccharide, and a step of purifying and drying thereaction product.

In the solution preparing step, the polysaccharide is added to asolvent, and heated at 60 to 120° C., so that the polysaccharide isdissolved in an aprotic polar solvent.

Consequently, the acid group-containing polysaccharide to beactive-esterified should preferably be one among the above listedpolysaccharides which is soluble in an aprotic polar solvent at atemperature in the range of 60° C. to 120° C. To be concrete, thepolysaccharide to be used for the reaction for introduction ofelectrophilic group should preferably be one in which the carboxy groupor the acid type carboxymethyl group is, from the standpoint ofsolubility in the aprotic polar solvent. The term “acid type” means thatthe species of counter cation of the carboxy group or the carboxymethylgroup is proton. The polysaccharide which has an acid type carboxy groupis referred to as acid type (starting) polysaccharide. For example,pectin as a polysaccharide having a carboxy group is referred to as acidtype pectin. Carboxymethyl dextran having acid type carboxymethyl groupis referred to as acid type carboxymethyl (CM) dextran or acid type CMdextran. The term “acid type” has the same meaning as the “non-salttype” mentioned above in the sense that the species of counter cation isproton, and is in the form of “non-salt”.

The “aprotic polar solvent” denotes a polar solvent which does not haveproton capable of forming hydrogen bond with a nucleophilic agent havingan electrically positive functional group. The aprotic polar solventthat can be used in the method of the present invention is notspecifically restricted; it includes, for example, dimethylsulfoxide(DMSO), N,N-dimethylformamide, N-methyl-2-pyrolidone,N,N-dimethylacetamide, and 1,3-dimethyl-2-imidazoline. Of theseexamples, dimethylsulfoxide may be used preferably because of itsdissolibility to polysaccharide.

In the reaction step, to the solution of acid type polysaccharide isadded the electrophilic group-introducing agent and thedehydrating-condensing agent so as to active-esterify the carboxy groupand/or the carboxymethyl group in the polysaccharide. The reactiontemperature for active-esterification is not specifically restricted; itis preferably 0° C. to 70° C., more preferably 20° C. to 40° C. Thereaction time varies depending on the reaction temperature; it isusually 1 to 48 hours, preferably 12 to 24 hours.

The “electrophilic group-introducing agent” refers to a regent whichintroduces an electrophilic group into the carboxy group or thecarboxyalkyl group, thereby converting it into an active ester group.The electrophilic group-introducing agent is not specificallyrestricted; it may be a compound for introduction of active ester, whichis generally used for peptide synthesis. It includes, for example,N-hydroxylamine-based compound for introduction of active ester. TheN-hydroxylamine-based compound for introduction of active ester is notspecifically restricted; it includes, for example, N-hydroxysuccinimide,N-hydroxynorbornene-2,3-dicarboxylic imide, ethyl ester of2-hydroxyimino-2-cyanoacetate, 2-hydroxyimino-2-cyanoacetic amide, andN-hydroxypiperidine. Of these examples, N-hydroxysuccinimide isdesirable because it is commercially available and has been widely usedin the field of peptide synthesis.

The “dehydrating-condensing agent” is one that withdraws one watermolecule, dehydrates in other word, which occurs by condensation betweenthe carboxy group or carboxyalkyl group and the electrophilicgroup-introducing agent when the carboxy group or carboxyalkyl group isconverted into the active ester group by the electrophilicgroup-introducing agent, thereby forming an ester linkage between them.The dehydrating-condensing agent is not specifically restricted; itincludes, for example, 1-ethyl-3-dimethylaminopropylcarbodiimidehydrochloride (EDC.HCl) and1-cyclohexyl-(2-morphonyl-4-ethyl)-carbodiimide-meso-p-toluenesulfonate.Of these examples, 1-ethyl-3-dimethylaminopropylcarbodiimidehydrochloride (EDC.HCl) is desirable because it is commerciallyavailable and has been widely used in the field of peptide synthesis.

The purifying step comprises ordinary reprecipitation (from reactionliquid), filtration, and/or washing, which are carried out after thereaction is complete. By the purifying step, was removed theelectrophilic group-introducing agent and dehydrating-condensing agentremaining unreacted and by-products, thereby was obtained thepolysaccharide derivative of the present invention.

The drying step may be carried out by a commonly used method to remove awashing solvent from the polysaccharide derivative obtained in thepurifying step.

According to the present invention, the amount of the active ester groupin the polysaccharide derivative which is eventually obtained shouldpreferably be 0.1 to 2 mmol/g, as mentioned above. In theabove-mentioned steps, it is possible to control the amount of activeester group to be introduced into the carboxy group of the startingpolysaccharide for active esterification so that the desiredpolysaccharide derivative is obtained.

In order to control the amount of active ester group to be introduced,it is possible to adjust the mixing amounts of the electrophilicgroup-introducing agent and the dehydrating condensation agent in theabove-mentioned reaction steps. More specifically, the additioncondition that the ratio (Z/X) of the number of moles (Z mmol) of thedehydrating condensation agent to the number of moles (X mmol) of totalcarboxy group in the polysaccharide is 0.1<Z/X<50 is preferably used. Ifthe ratio Z/X is smaller than 0.1, the reaction efficiency is lowbecause of a small amount of the dehydrating condensation agent added.Therefore, it is difficult to achieve a desired active estergroup-introducing ratio. If the ratio Z/X is larger than 50, a highactive ester group-introducing ratio is achieved because of a largeramount of the dehydrating condensation agent but the resultingpolysaccharide is hardly soluble in water.

The number of moles of the electrophilic group-introducing agent (Y mol)to the number of moles of total carboxy group in the polysaccharide isnot specifically restricted so long as the electrophilicgroup-introducing agent and the dehydrating condensation agent are addedin amounts equal to or larger than the reaction amounts corresponding tothe active ester group introducing-ratio. However, the additioncondition satisfying 0.1<Y/X<100 is preferably used.

The polysaccharide derivative according to the present invention usuallyretains the hydroxy group of the glucopyranose ring in its skeletonmolecule even after the active ester group have been introduced therein.Therefore, the polysaccharide derivative has the activehydrogen-containing group. The active hydrogen-containing group in themolecule are not limited thereto and the polysaccharide may additionallyhave active hydrogen-containing groups introduced in the molecule asrequired. In this case, the polysaccharide derivative may have one kindor more than one kind of active hydrogen-containing group.

In addition to the above-mentioned active ester group and activehydrogen-containing group, the polysaccharide derivative of the presentinvention may contain any known elements, atomic groups, or otherfunctional groups in the amounts that do not adversely affect the meritsof the present invention.

Examples of the functional group include halogen elements such asfluorine, chlorine, bromine, and iodine, carboxy group, carboxyalkylgroups such as carboxymethyl group, carboxyethyl group, carboxypropylgroup, and carboxyisoproyl group, silyl group, alkylenesilyl group,alkoxysilyl group, and phosphate group. These functional groups may beused alone or in combination of two or more.

The active ester group-introducing ratio (%) can be represented bymultiplying by 100 a ratio (AE/TC) of the molar amount of the activeester group in the resulting polysaccharide derivative (AE) to the molaramount of the carboxy group in the starting polysaccharide for theactive esterification and the molar amount of the carboxymethyl group(hereinafter referred to as total carboxy group (TC).

The active ester group-introducing ratio can be determined by, forexample, the method described in Biochemistry vol. 14, No. 7 (1975), pp.1535 to 1541.

In particular, the polysaccharide derivative may include residualcarboxy group and/or carboxymethyl group from the startingpolysaccharide has when the active ester group are introduced at anactive ester group-introducing ratio of less than 100%.

The term “crosslinked structure” means a three-dimensional networkstructure of the molecular chains of the polysaccharide derivative whichis formed through covalent bond within one molecular chain and/orbetween two or more molecular chains of the polysaccharide derivative ofthe present invention. The active ester group and the activehydrogen-containing group may be bound together within one molecularchain through crosslinking, but crosslinking may be formed throughcovalent bond between two or more molecules. The polysaccharidederivative of the present invention is water-soluble before it iscrosslinked. However, when the reaction progresses, it forms acrosslinked structure, which reduces flowability to producewater-insoluble aggregated matrix (water-containing gel), whereby acrosslinked polysaccharide is formed. The polysaccharide derivative ofthe present invention is a self-crosslinkable polysaccharide. The term“self-crosslinkable” is defined as the property of forming thecrosslinked structure through covalent bond within its own molecule orbetween molecular chains without particularly using other crosslinkingagent.

The polysaccharide derivative of the present invention exhibits not onlythe self-crosslinking property in which intramolecular activehydrogen-containing group is involved but also the adhesiveness to thesurface of living organism through reaction of the activehydrogen-containing group on the surface of the living organism with theactive ester group, if it is applied to the surface of the livingorganism. This mode of use is desirable for the polysaccharidederivative of the present invention. The self-crosslinking may also beformed when the polysaccharide derivative is applied to the surface ofthe living organism.

According to the present invention, the active hydrogen-containing groupinvolved in the reaction with the active ester group is not specificallyrestricted so long as it is a group that may form covalent bond throughreaction with the active ester group under specified conditions in thepresent invention. Any active hydrogen-containing group can also be usedin the present invention. Specific examples include hydroxy group, aminogroup, and thiol group. Examples of the amino group include primaryamino group and secondary amino group. Of these, the case where theactive hydrogen-containing group is hydroxy group or primary amino groupis preferable because of excellent reactivity with the active estergroup and short time for crosslinking to gelation.

The method of crosslinking the polysaccharide derivative of the presentinvention refers to a method of forming covalent bond through reactionbetween the active ester group and the active hydrogen-containing group.Specific examples include a method in which the polysaccharidederivative of the present invention is crosslinked under alkalineconditions in the presence of moisture such as water, water vapor, orwater-containing solvent, and a method in which a pH adjusting agent isadded to a polysaccharide derivative solution for crosslinking.

To be more specific, it is possible to crosslink the polysaccharidederivative in the presence of water at a pH of 7.5 to 12, preferably 9.0to 10.5. If the pH of the water is lower than 7.5, self-crosslinkingproperty is low and a sufficient degree of crosslinking is not obtained.On the other hand, even if crosslinking progresses at a pH of 12 ormore, this is not appropriate considering the physiological conditions.

The term “alkaline conditions” used in the present invention means thecondition under which moisture having a pH of 7.5 or more is present.The “alkaline conditions” is not specifically restricted in temperature;but the temperature can be set for example in a range of 10° C. to 40°C., because heat does not greatly contribute to the crosslinking of thecrosslinkable polysaccharide derivative of the present invention.

The term “brought into contact with water under alkaline conditions”means that the polysaccharide derivative is brought into contact withmoisture in any form under alkaline conditions to place it underalkaline conditions. When the polysaccharide derivative is in powderform, it is possible to add water adjusted to alkaline conditions or toadd water to a mixture of the polysaccharide derivative powder with a pHadjusting agent. When the polysaccharide derivative is in the form ofaqueous solution, it is possible to add water previously adjusted toalkaline conditions or to add a pH adjusting agent. These operationsplace the polysaccharide derivative in an alkaline environment, and thecrosslinking reaction starts. In other words, the polysaccharidederivative starts crosslinking upon contact with moisture under alkalineconditions and the crosslinking progresses. Therefore, the mixture ofthe polysaccharide derivative and water under alkaline conditions mayhave a pH under alkaline conditions, but the alkaline condition is notobligatory. The polysaccharide derivative begins to crosslink uponcontact with water under alkaline conditions, but crosslinking reactiondoes not substantially start or proceed by exposure to UV light or byheating.

In the present invention, the polysaccharide derivative mentioned abovecan be provided as a crosslinkable material only composed of thepolysaccharide derivative for its self-crosslinking property. Acrosslinkable material in the form of a composition composed of acombination with other components can also be provided. Depending on thetype, other components may be added to form a composition in contactwith the polysaccharide derivative or may not be in contact therewithuntil they are mixed together before use.

The polysaccharide derivative can be provided in the form of powder orsheet. In other words, the polysaccharide derivative in powder form canbe obtained by crushing or grinding the polysaccharide derivativeprepared by the above-mentioned synthesis and optionally adjusting theparticle size. There is no particular limitation to reduce the particlesize, but freeze grinding, milling and/or classification may beperformed after crushing or grinding, the particle size can also beadjusted by sieving to have any particle size distribution. The averageparticle size is not specifically restricted; however, it shouldpreferably be tens of nanometers to hundreds of micrometers. Theresulting powder may be made into paste or aerosol by a commonly usedmethod.

The polysaccharide derivative in sheet form can be prepared by asolution preparing step in which the polysaccharide derivative isdissolved in water and a drying step in which the solution is spread outinto a desired shape and heat-dried or freeze-dried. To be morespecific, the polysaccharide derivative in sheet form can be obtained bypreparing an aqueous solution in which the polysaccharide derivative isdissolved and freeze-drying the aqueous solution. When thepolysaccharide derivative in sheet form is prepared, it is desirablethat the water used for preparing the aqueous solution have a pH of 3.0to 7.5. If the water has a pH of not more than 3.0, the resulting sheetexhibits strong acidity. If the water has a pH of 7.5 or more, theactive ester group is often released. The heat-dried sheet can beobtained by spreading the aqueous solution on a substrate and thenheat-drying it at 30° C. to 110° C. If necessary, heat-drying may beaccomplished under reduced pressure. The freeze-dried sheet can beobtained by freezing the aqueous solution and then drying it in itsfrozen state. If necessary, freeze-drying may be accomplished by usingan ordinary freeze-drier.

The present invention provides a crosslinkable polysaccharidecomposition containing the polysaccharide derivative (A) and the pHadjusting agent (B) as a composition containing the above-mentionedpolysaccharide derivative (A). This composition can be used as anadhesive or glue.

The pH adjusting agent (B) may be supplied without being mixed or afterhaving previously been mixed. The period when the pH adjusting agent (B)is mixed is not specifically restricted, but the period is appropriatelyselected from before use and during use. The composition including thepolysaccharide derivative (A) and the pH adjusting agent (B) mayoptionally contain any other substances. Such other substances may ormay not be mixed with the polysaccharide derivative.

The pH adjusting agent (B) used in the present invention denotes anaqueous solution, a water-containing solvent, or salt (powder) foradjusting the polysaccharide derivative or the crosslinkablepolysaccharide composition of the present invention to a pH of 7.5 to12. The pH adjusting agent (B) is not specifically restricted. Specificexamples include sodium hydrogencarbonate in the form of aqueoussolution or powder, phosphate buffer (disodium hydrogenphosphate andpotassium dihydrogenphosphate), and acetic acid-ammonia buffer. Ofthese, sodium hydrogencarbonate can be suitably used from the viewpointof safety in that its 7% aqueous solution (pH 8.3) of sodiumhydrogencarbonate is used as a pH adjusting agent for medical purposesin the form of intravenous injection.

The above-mentioned composition may be in two-component type, with onebeing an aqueous solution containing 1 to 80% (w/v) of thepolysaccharide derivative and the other being water adjusted to a pH of7.5 to 10.5.

The two components can be mixed together before use to obtain an aqueoussolution having a final polysaccharide derivative concentration of 0.1to 60% (w/v). Alternatively, it is possible to add a salt of the pHadjusting agent (B) to an aqueous solution having a polysaccharidederivative concentration of 1 to 80% (w/v) before use and dissolve thesalt therein to obtain a mixture having a final polysaccharidederivative concentration of 0.1 to 80% (w/v). Mixing may be accomplishedin a conventional mixing method, but it is preferable to mix to ahomogenous state. The resulting mixture should be homogenous enough topermit a desired reaction to proceed.

The present invention also provides the crosslinkable polysaccharidecomposition (also abbreviated as polysaccharide composition) whichcontains the polysaccharide derivative (A) and another polymer (C). Thepolymer (C) is used to adjust the hardness and other properties ofhydrogel which is formed when the polysaccharide composition iscrosslinked. The polysaccharide derivative (A) may be used alone or incombination of two or more. The pH adjusting agent (B) may also becontained in the composition.

The polymer (C) is not specifically restricted, but it is preferable touse one which has more than one primary amino group, thiol group, orhydroxy group in one molecule. Specific examples of the polymer (C)include polyalkylene glycol derivative, polypeptide, polysaccharide, anda derivative thereof. The content of the polymer (C) in thepolysaccharide composition of the present invention is not specificallyrestricted, but it should preferably be 5 to 50 wt % based on the totalamount of the polysaccharide composition. The polymer (C) can be usedalone or in combination of two or more.

The polyalkylene glycol derivative mentioned above includes, forexample, polyethylene glycol (PEG) derivative, polypropylene glycolderivative, polybutylene glycol derivative, and polypropyleneglycol-polyethylene glycol block copolymer derivative and randomcopolymer derivative. The polyethylene glycol derivative has the mainpolymer backbone of ethylene glycol, diglycerol, pentaerythritol, orhexaglycerol. The polyalkylene glycol derivative should preferably havea molecular weight of 100 to 50,000, more preferably 1,000 to 20,000.

The polyethylene glycol mentioned above is not specifically restricted;it includes, for example, ethylene glycol-type polyethylene glycolderivative having a thiol group on both terminals and having aweight-average molecular weight of 1,000, 2,000, 6,000, or 10,000,ethylene glycol-type polyethylene glycol derivative having an aminogroup on both terminals and having a weight-average molecular weight of1,000, 2,000, 6,000, or 10,000, trimethylol ethane-type polyethyleneglycol derivative having a thiol group on three terminals and having aweight-average molecular weight of 5,000 or 10,000, trimethylolethane-type polyethylene glycol derivative having an amino group onthree terminals and having a weight-average molecular weight of 5,000 or10,000, diglycerol-type polyethylene glycol derivative having a thiolgroup on four terminals and having a weight-average molecular weight of5,000, 10,000, or 20,000, diglycerol-type polyethylene glycol derivativehaving an amino group on four terminals and having a weight-averagemolecular weight of 5,000, 10,000, or 20,000, pentaerythritol-typepolyethylene glycol derivative having a thiol group on four terminalsand having a weight-average molecular weight of 10,000 or 20,000,pentaerythritol-type polyethylene glycol derivative having an aminogroup on four terminals and having a weight-average molecular weight of10,000 or 20,000, hexaglycerol-type polyethylene glycol derivativehaving a thiol group on eight terminals and having a weight-averagemolecular weight of 10,000 or 20,000, and hexaglycerol-type polyethyleneglycol derivative having an amino group on eight terminals and having aweight-average molecular weight of 10,000 or 20,000.

The term “weight-average molecular weight” is a numerical value thatrepresents the average molecular weight of a polymer. Since a polymer isa mixture of molecules having the same main structural units butdiffering in the length of molecules (or chains), it has a distributionof molecular weight according to the lengths of molecules. To indicatethe molecular weight, the average molecular weight is used. The averagemolecular weight may be represented in terms of weight-average molecularweight, number-average molecular weight or the like. Here, theweight-average molecular weight is used. In the present invention, thevalue (100%) of the weight-average molecular weight embraces the onewhose upper limit is 110% and whose lower limit is 90%. The polyethyleneglycol derivative may be prepared by the process mentioned in Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications,compiled by J. Milton Harris, issued by Plenum Press, NY (1992), Chapter22. It may also be chemically modified such that it contains one or morethan one primary amino group or thiol group. Also, the polyethyleneglycol derivative is commercially available from NOF Corporation underthe trade names of Sunbrite HGEO-20TEA, Sunbrite PTE-10TSH or the like.

The polypeptide mentioned above is not specifically restricted; itincludes, for example, collagen, gelatin, albumin, and polylysine. Thepolysaccharide is not specifically restricted; it includes, for example,pectin, hyaluronic acid, chitin, chitosan, carboxymethylchitin,carboxymethylchitosan, chondroitin sulfate, keratin sulfate, keratosulfate, and heparin, and derivatives thereof.

The polysaccharide composition of the present invention, which iscomposed of the polysaccharide derivative (A) (active esterifiedpolysaccharide) and the polymer (C), should preferably have thefollowing combination of (A) and (C). The form (sheet, powder, liquid)for each combination will be properly selected with reference toExamples mentioned later.

Combination of active esterified pectin with at least one polymer (C)selected from the group consisting of ethylene glycol-type PEGderivative having a thiol group on both terminals, ethylene glycol-typePEG derivative having an amino group on both terminals, trimethylolethane-type PEG derivative having a thiol group on three terminals,trimethylol ethane-type PEG derivative having an amino group on threeterminals, pentaerythritol-type PEG derivative having a thiol group onfour terminals, pentaerythritol-type PEG derivative having an aminogroup on four terminals, hexaglycerol-type PEG derivative having a thiolgroup on eight terminals, hexaglycerol-type PEG derivative having anamino group on eight terminals, albumin, gelatin, collagen, polylysine,pectin, chitosan, chitin, and carboxymethyl (CM) chitin.

Combination of active esterified CM dextran with at least one polymer(C) selected from the group consisting of ethylene glycol-type PEGderivative having a thiol group on both terminals, ethylene glycol-typePEG derivative having an amino group on both terminals, trimethylolethane-type PEG derivative having a thiol group on three terminals,trimethylol ethane-type PEG derivative having an amino group on threeterminals, pentaerythritol-type PEG derivative having a thiol group onfour terminals, pentaerythritol-type PEG derivative having an aminogroup on four terminals, hexaglycerol-type PEG derivative having a thiolgroup on eight terminals, hexaglycerol-type PEG derivative having anamino group on eight terminals, albumin, gelatin, collagen, polylysine,pectin, chitosan, chitin, and CM chitin.

Combination of active esterified CM pullulan with at least one polymer(C) selected from the group consisting of ethylene glycol-type PEGderivative having a thiol group on both terminals, ethylene glycol-typePEG derivative having an amino group on both terminals, trimethylolethane-type PEG derivative having a thiol group on three terminals,trimethylol ethane-type PEG derivative having an amino group on threeterminals, pentaerythritol-type PEG derivative having a thiol group onfour terminals, pentaerythritol-type PEG derivative having an aminogroup on four terminals, hexaglycerol-type PEG derivative having a thiolgroup on eight terminals, hexaglycerol-type PEG derivative having anamino group on eight terminals, albumin, gelatin, collagen, polylysine,pectin, chitosan, chitin, and CM chitin.

Combination of active esterified CM hydroxyethyl starch with at leastone polymer (C) selected from the group consisting of ethyleneglycol-type PEG derivative having a thiol group on both terminals,ethylene glycol-type PEG derivative having an amino group on bothterminals, trimethylol ethane-type PEG derivative having a thiol groupon three terminals, trimethylol ethane-type PEG derivative having anamino group on three terminals, pentaerythritol-type PEG derivativehaving a thiol group on four terminals, pentaerythritol-type PEGderivative having an amino group on four terminals, hexaglycerol-typePEG derivative having a thiol group on eight terminals,hexaglycerol-type PEG derivative having an amino group on eightterminals, albumin, gelatin, collagen, polylysine, pectin, chitosan,chitin, and CM chitin.

Combination of active esterified pectin with at least one polymer (C)selected from the group consisting of ethylene glycol-type PEGderivative having a thiol group on both terminals and having aweight-average molecular weight of 1,000, 2,000, 6,000, or 10,000,ethylene glycol-type PEG derivative having an amino group on bothterminals and having a weight-average molecular weight of 1,000, 2,000,6,000, or 10,000, trimethylol ethane-type PEG derivative having a thiolgroup on three terminals and having a weight-average molecular weight of5,000 or 10,000, trimethylol ethane-type PEG derivative having an aminogroup on three terminals and having a weight-average molecular weight of5,000 or 10,000, diglycerol-type PEG derivative having a thiol group onfour terminals and having a weight-average molecular weight of 5,000,10,000, or 20,000, diglycerol-type PEG derivative having an amino groupon four terminals and having a weight-average molecular weight of 5,000,10,000, or 20,000, pentaerythritol-type PEG derivative having a thiolgroup on four terminals and having a weight-average molecular weight of10,000 or 20,000, pentaerythritol-type PEG derivative having an aminogroup on four terminals and having a weight-average molecular weight of10,000 or 20,000, hexaglycerol-type PEG derivative having a thiol groupon eight terminals and having a weight-average molecular weight of10,000 or 20,000, and hexaglycerol-type PEG derivative having an aminogroup on eight terminals and having a weight-average molecular weight of10,000 or 20,000.

Combination of active esterified CM dextran with at least one polymer(C) selected from the group consisting of ethylene glycol-type PEGderivative having a thiol group on both terminals and having aweight-average molecular weight of 1,000, 2,000, 6,000, or 10,000,ethylene glycol-type PEG derivative having an amino group on bothterminals and having a weight-average molecular weight of 1,000, 2,000,6,000, or 10,000, trimethylol ethane-type PEG derivative having a thiolgroup on three terminals and having a weight-average molecular weight of5,000 or 10,000, trimethylol ethane-type PEG derivative having an aminogroup on three terminals and having a weight-average molecular weight of5,000 or 10,000, diglycerol-type PEG derivative having a thiol group onfour terminals and having a weight-average molecular weight of 5,000,10,000, or 20,000, diglycerol-type PEG derivative having an amino groupon four terminals and having a weight-average molecular weight of 5,000,10,000, or 20,000, pentaerythritol-type PEG derivative having a thiolgroup on four terminals and having a weight-average molecular weight of10,000 or 20,000, pentaerythritol-type PEG derivative having an aminogroup on four terminals and having a weight-average molecular weight of10,000 or 20,000, hexaglycerol-type PEG derivative having a thiol groupon eight terminals and having a weight-average molecular weight of10,000 or 20,000, and hexaglycerol-type PEG derivative having an aminogroup on eight terminals and having a weight-average molecular weight of10,000 or 20,000.

Combination of active esterified pullulan with at least one polymer (C)selected from the group consisting of ethylene glycol-type PEGderivative having a thiol group on both terminals and having aweight-average molecular weight of 1,000, 2,000, 6,000, or 10,000,ethylene glycol-type PEG derivative having an amino group on bothterminals and having a weight-average molecular weight of 1,000, 2,000,6,000, or 10,000, trimethylol ethane-type PEG derivative having a thiolgroup on three terminals and having a weight-average molecular weight of5,000 or 10,000, trimethylol ethane-type PEG derivative having an aminogroup on three terminals and having a weight-average molecular weight of5,000 or 10,000, diglycerol-type PEG derivative having a thiol group onfour terminals and having a weight-average molecular weight of 5,000,10,000, or 20,000, diglycerol-type PEG derivative having an amino groupon four terminals and having a weight-average molecular weight of 5,000,10,000, or 20,000, pentaerythritol-type PEG derivative having a thiolgroup on four terminals and having a weight-average molecular weight of10,000 or 20,000, pentaerythritol-type PEG derivative having an aminogroup on four terminals and having a weight-average molecular weight of10,000 or 20,000, hexaglycerol-type PEG derivative having a thiol groupon eight terminals and having a weight-average molecular weight of10,000 or 20,000, and hexaglycerol-type PEG derivative having an aminogroup on eight terminals and having a weight-average molecular weight of10,000 or 20,000.

Combination of active esterified CM hydroxyethyl starch with at leastone polymer (C) selected from the group consisting of ethyleneglycol-type PEG derivative having a thiol group on both terminals andhaving a weight-average molecular weight of 1,000, 2,000, 6,000, or10,000, ethylene glycol-type PEG derivative having an amino group onboth terminals and having a weight-average molecular weight of 1,000,2,000, 6,000, or 10,000, trimethylol ethane-type PEG derivative having athiol group on three terminals and having a weight-average molecularweight of 5,000 or 10,000, trimethylol ethane-type PEG derivative havingan amino group on three terminals and having a weight-average molecularweight of 5,000 or 10,000, diglycerol-type PEG derivative having a thiolgroup on four terminals and having a weight-average molecular weight of5,000, 10,000, or 20,000, diglycerol-type PEG derivative having an aminogroup on four terminals and having a weight-average molecular weight of5,000, 10,000, or 20,000, pentaerythritol-type PEG derivative having athiol group on four terminals and having a weight-average molecularweight of 10,000 or 20,000, pentaerythritol-type PEG derivative havingan amino group on four terminals and having a weight-average molecularweight of 10,000 or 20,000, hexaglycerol-type PEG derivative having athiol group on eight terminals and having a weight-average molecularweight of 10,000 or 20,000, and hexaglycerol-type PEG derivative havingan amino group on eight terminals and having a weight-average molecularweight of 10,000 or 20,000.

The ratio of the polysaccharide derivative (A) (SD) to the polymer (C)(AP) should preferably be SD/AP=20/80 to 98/2 (w/w). If the amount ofthe polymer (C) is more than 80 wt %, the polysaccharide derivative (A)hardly exhibits its self-crosslinking property. If the amount of thepolymer (C) is less than 2 wt %, it will be difficult to control thehardness and other properties of the hydrogel to be obtained eventually.

The polysaccharide composition of the present invention may beincorporated with various kinds of known additives in an amount notharmful to the effect of the present invention. The additives are notspecifically restricted; they include cure catalyst, filler,plasticizer, softener, stabilizer, dehydrizer, coloring agent, anti-sagagent, thickener, property adjusting agent, reinforcing agent,thixotropic agent, age resistor, flame retardant, antioxidant, UV lightabsorber, pigment, solvent, carrier, shaping agent, antiseptic, binder,antioxidant, swelling agent, isostatic agent, solubilizing agent,preservative, buffer solution, and diluent. These additives may be usedalone or in combination with one another.

The polysaccharide composition mentioned above may be provided in theform of sheet, powder, or liquid. The polysaccharide composition inpowder form may be prepared by mixing the polysaccharide derivative (A)in powder form mentioned above with the polymer (C) in powder form. Thepolysaccharide composition in powder form mentioned may also be mixedwith a salt of pH adjusting agent in powder form to give thepolysaccharide composition containing a salt of pH adjusting agent.

The above-mentioned polysaccharide composition in powder form or theabove-mentioned polysaccharide composition in powder form containing asalt of pH adjusting agent may be made into granules by granulation. Theabove-mentioned polysaccharide composition in powder form or theabove-mentioned polysaccharide composition in powder form containing asalt of pH adjusting agent may also be made into sheet or plate bycompression. The polysaccharide composition in sheet form may beobtained by attaching the polymer (C) in powder form or by impregnatingwith the polymer (C) by coating, to the heat-dried or freeze-dried sheetof the above-mentioned polysaccharide derivative (A). The term“impregnating” means that the surface of the sheet is impregnated withthe polymer (C) so that the surface of the sheet is covered with thepolymer (C). In the case where the sheet is of porous structure,“impregnating” means that the polymer (C) covers the sheet surface andthe pores' internal surface inside of the sheet.

The polysaccharide composition may be of two-pack type, which consistsof an aqueous solution of the polysaccharide derivative (A) and anaqueous solution of the polymer (C). Upon mixing of these two aqueoussolutions, there is obtained a hydrogel consisting of the polysaccharidederivative (A) and the polymer (C). The aqueous solution of thepolysaccharide derivative (A) should preferably have a concentration of1 to 80% (w/v), and the aqueous solution of the polymer (C) shouldpreferably have a concentration of 1 to 80% (w/v). The polymer (C) maybe dissolved in water adjusted to pH 7.5 to 10.5 or in pure water orbuffer solution which is incorporated with a salt of pH adjusting agentat the time of mixing. After the aqueous solutions of the polysaccharidederivative (A) and the polymer (C) have been mixed together, the totalconcentration of the polysaccharide derivative (A) and the polymer (C)in the mixed solution should preferably be 0.1 to 80% (w/v).

The polysaccharide composition in sheet crosslinks when used in thepresence of water. Water may be the above-mentioned pH adjusting agent.The pH adjusting agent should preferably be an aqueous solution adjustedto pH 7.5 to 10.5. The pH adjusting agent in power form may be attachedto the polysaccharide composition in sheet form.

The polysaccharide composition in sheet form can be prepared bydissolving the polysaccharide derivative (A) in water, spreading theresulting aqueous solution into a desired shape, drying the spreadsolution, and impregnating the polymer (C) to the polysaccharidederivative (A) in sheet form thus obtained. The impregnating step may beaccomplished by dipping the sheet in the polymer (C) and a solutioncontaining a non-aqueous volatile organic solvent, followed by drying.In this way it is possible to impregnate the polymer (C) withoutimpairing the surface shape of the polysaccharide derivative (A) insheet form. Incidentally, the term “non-aqueous volatile organicsolvent” means any water-incompatible volatile organic solvent. Thenon-aqueous volatile organic solvent is not specifically restricted; itincludes chloroform and dichloromethane.

The polysaccharide derivative of the present invention and thepolysaccharide composition containing it may be used in a desired formas a medical treatment material. The term “medical treatment material”means any substance which is composed of components safe and acceptable(with a low level of toxicity) to the living body when used in theliving body. It may or may not be biodegradable in the living body. Itshould preferably be biodegradable in the living body. For example, itmay be used for hemostasis, adhesion, sealing, and/or fixing of thetissue or organ during operation. The medical treatment material is notspecifically restricted in form; it may be in the form of sheet, powder,paste, or aerosol.

The medical treatment material may be used in the form of mixture withthe above-mentioned pH adjusting agent. The medical treatment materialmay be mixed with the pH adjusting agent previously or in situ at thetime of use. The medical treatment material may be applied to thedesired spot after incorporation with an aqueous solution of the pHadjusting agent or the like at the time of use.

As in the case of the polysaccharide composition of the presentinvention, the medical treatment material may also be incorporated withvarious kinds of known additives, preferably compatible with the livingbody, in an amount not harmful to the effect of the present invention.The additives are not specifically restricted; they include carrier,shaping agent, antiseptic, stabilizer, binder, antioxidant, swellingagent, isostatic agent, solubilizing agent, preservative, buffersolution, and diluent. These additives may be used alone or incombination of two or more.

The additives are exemplified by water, physiological saline, organicsolvent for medicinal use, gelatin, collagen, polyvinyl alcohol,polyvinyl pyrrolidone, carboxyvinyl polymer, sodiumcarboxymethylcellulose, sodium polyacrylate, sodium alginate,water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, tragacanth, casein,agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum,paraffin, stearyl alcohol, stearic acid, human blood serum albumin(HSA), mannitol, sorbitol, lactose, PBS, nonionic surfactant,biodegradable polymer, serum-free culture medium, surfactants acceptablefor use as pharmaceutical additive, and buffer solution with aphysiological pH suitable for living bodies.

The carrier may be properly selected alone or in combination from theforegoing according to the wound for application. However, the carriersare not restricted to those mentioned above. The medical treatmentmaterial may be prepared into gel or aerosol (in combination with anadequate propellant).

The medical treatment material may be provided in the form of kitcontaining the above-mentioned pH adjusting agent for convenience at thetime of use. The medical treatment material in the form of kit includesthe polysaccharide derivative (A), the polysaccharide composition,and/of the pH adjusting agent, which are not mixed together. They arecontained all together or separately in one package. The medicaltreatment material may contain any other constituents which can be usedas medical treatment material.

The kit may contain the polysaccharide derivative or polysaccharidecomposition in the form of powder, sheet, or aqueous solution which mayor may not contain the pH adjusting agent in the form of aqueoussolution or powder.

The medical treatment material may be used as a hemostatic and/oradhesive. The hemostatic is used to stop bleeding in a living body. Tothis end, the medical treatment material is applied to the bleedingwound. The medical treatment material is applied to a target portion andcovers the bleeding portion as required to stop bleeding and thusachieve the hemostatic effect. The adhesive is used to bond at least onepart of the living tissue or organ to the other part. To this end, themedical treatment material is applied to the desired spot and allowed tostand under pressure (or without pressure) for a certain period of time.Upon the above process, it is possible to use a fixing tool or the like.

The polysaccharide derivative or the polysaccharide composition, whichis in the form of power, sheet, or aqueous solution containing or notcontaining the pH adjusting agent in the form of aqueous solution orpowder, may be contained in the hemostatic and/or adhesive.

The medical treatment material of the present invention is used as ahemostatic and/or adhesive. Consequently, the present invention offers amethod of stopping bleeding and/or bonding living organism by briningthe medical treatment material into contact with a desired spot in thepresence of water. The object is achieved by spraying, filling, orcoating the medical treatment material in powder form. For the medicaltreatment material in sheet form, the object is achieved by pasting,filling, covering, pressing, and allowing to stand. For the medicaltreatment material in liquid form, the object is achieved by applying,spraying, dropping, and rubbing. In this way it is possible to achievehemostasis and/or adhesion.

The polysaccharide derivative of the present invention has an activeester group and an active hydrogen-containing group in one molecularchain, so that they react with each other to form a covalent bond,thereby forming the crosslinked structure. When used as an adhesive, itproduces sufficient tissue bond strength that meets the clinicalrequirements and it avoids risk of infection because it does not use anymaterial originating from living tissue but it is based on natural orartificial polysaccharide for the backbone. Its components or theirdecomposition products have a low level of toxicity and are capable ofbiodegradation and bioabsorption because the backbone is formed frompolysaccharide. The polysaccharide derivative of the present inventiondoes not need complex steps for preparation at the time of use. It canbe prepared readily and simply without requiring special apparatus atthe time of use. The polysaccharide derivative may be provided alone oras the polysaccharide composition; therefore, it will find a largevariety of uses. Since the polysaccharide composition of the presentinvention employs the polysaccharide derivative of the present inventionhaving the property described above, it does not impair thecharacteristic properties of the present invention.

The polysaccharide derivative and polysaccharide composition of thepresent invention can be fabricated into powder, sheet, granules, or anyother forms. Therefore, they can be used in various ways according tothe object of their use. The polysaccharide derivative andpolysaccharide composition of the present invention can be producedsimply by mixing with heating necessary reagents, without requiringspecial apparatus. Owing to the above-mentioned characteristicproperties, the polysaccharide derivative and polysaccharide compositionof the present invention are suitable for use as the medical treatmentmaterial such as hemostatic agent and adhesive.

EXAMPLES (I) Preparation of Starting Polysaccharide

An acid group-containing polysaccharide that is a polysaccharide havingan acid-type carboxy group or an acid-type carboxymethyl group wasprepared as a starting polysaccharide to be a starting material for theactive esterified polysaccharide (polysaccharide derivative) of thepresent invention.

(1) Preparation of Acid-type Pectin

Five grams of pectin (GENU Pectin USP-H from CP Kelco) was suspended in500 mL of 90 vol % methanol aqueous solution (100% methanol from WakoPure Chemical Industries, Ltd.). The resulting suspension was adjustedto pH 1.0 with 20% hydrochloric acid (hydrochloric acid from Wako PureChemical Industries, Ltd.). After stirring at 25° C. for 2 hours, thesuspension was filtered through a suction funnel to recover pectin. Therecovered pectin was washed with 2 liters of 80 vol % methanol aqueoussolution, and finally washed with 100% methanol, and vacuum dried.Consequently, an acid-type pectin was obtained.

(2) Preparation of Acid-type Hyaluronic Acid

An acid-type hyaluronic acid was prepared in the same way as in (I)-(1)except that the pectin used in (I)-(1) was replaced by sodium hyalronate(Hyaluronic acid FCH-150 from Kibun Food Inc.).

(3) Preparation of Acid-type Carboxymethyldextran A (Acid-type CMDextran A)

Twenty grams of dextran (Dextran T-40 from Amercham Biosciences, havinga weight-average molecular weight of 40,000) was dissolved in 75 mL ofpure water. To the resulting solution was added 50 mL of 45% (w/v)sodium hydroxide aqueous solution (sodium hydroxide from Wako PureChemical Industries, Ltd.). The resulting solution was stirred at 25° C.for 2 hours. Then, 75 mL of 40% (w/v) aqueous solution ofmonochloroacetic acid (monochloroacetic acid from Wako Pure ChemicalIndustries, Ltd.), and the resulting solution was stirred at 25° C. for18 hours. The reaction liquid was adjusted to pH 1.0 with 20%hydrochloric acid, followed by stirring at 25° C. for 2 hours. Thereaction liquid was added dropwise to 5 L of 90 vol % ethanol aqueoussolution (100% ethanol from Wako Pure Chemical Industries, Ltd.).Precipitates were recovered by filtration through a suction funnel. Therecovered precipitates were washed with 3 L of 90 vol % ethanol aqueoussolution, finally, washed with ethanol. Upon drying in vacuo, there wasobtained the acid-type CM dextran A.

(4) Preparation of Acid-type CM Dextran B

An acid-type CM dextran B was prepared in the same way as in (I)-(3)except that the dextran (20 g) used in (I)-(3) was replaced by dextranhaving a different molecular weight (Dextran T-500 from AmershamBiosciences, having a weight-average molecular weight of 500,000).

(5) Preparation of Acid-type Carboxymethyl Pullulan (Acid-type CMPullulan)

An acid-type CM pullulan was prepared in the same way as in (I)-(3)except that the dextran (20 g) used in (I)-(3) was replaced by 10 g ofpullulan (PI-20, from Hayashibarasha).

(II) Quantitative Determination of Carboxy Group or Carboxymethyl Group

Quantitative determination of carboxy group or carboxymethyl group wasperformed for the starting polysaccharide obtained in (I)-(1) to(I)-(5). 0.2 g (A g) of the starting polysaccharide was weighed, anddissolved in a mixed solution composed of 20 mL of aqueous solution ofsodium hydroxide (0.1 mol/L) and 10 mL of aqueous solution of methanol(80 vol %), followed by stirring at 25° C. for 3 hours. Three drops of1.0% (w/v) phenolphthalein solution in 90 vol % ethanol (as anindicator) were added to the solution (Phenolphthalein from Wako PureChemical Industries, Ltd.). An acid-base back titration was performed byusing 0.05 mol/L sulfuric acid, and the amount used of 0.05 mol/Lsulfuric acid (V1 mL) was determined. In blank test in the same wayexcept adding the starting polysaccharide, the amount of spent 0.05mol/L sulfuric acid (V0 mL) was determined. The amount (B mmol/g) ofcarboxy group and carboxymethyl group in the starting polysaccharide wascalculated from the following equation. Incidentally, both the 0.1 mol/Laqueous solution of sodium hydroxide and the 0.05 mol/L sulfuric acidhave a titer of 1.00. The results are shown in Table 1 below.B=(V ₀ −V ₁)×0.1÷A  (1)where,

A: mass of starting polysaccharide (g)

B: amount of carboxy group and carboxymethyl group (mmol/g)

TABLE 1 Acid-type starting Amount of carboxy group and polysaccharidecarboxymethyl group (mmol/g) Pectin 1.40 Hyaluronic acid 2.15 CM dextranA 1.01 CM dextran B 0.29 CM pullulan 1.28

(III) Preparation of Active Esterified Polysaccharide (PolysaccharideDerivative)

An active esterified polysaccharide (polysaccharide derivative) wasprepared by the above-mentioned active esterification reaction of theacid-type starting polysaccharide, in which DMSO as the reaction medium,N-hydroxysuccinimide (NHS) (from Wako Pure Chemical Industries, Ltd.) asthe electrophilic group-introducing agent, and1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) (from WakoPure Chemical Industries, Ltd.) as the dehydrating-condensing agent wereused.

Example 1 Preparation of Active Esterified Pectin

In 200 g of DMS was added 2.0 g of acid-type pectin (containing 1.40mmol/g of carboxy group) prepared in (I)-(1), and dissolved by stirringat 25° C. for 15 hours. The resulted solution was added 0.322 g (2.80mmol) of NHS and 0.536 g (2.80 mmol) of EDC, followed by stirring at 25°C. for 24 hours. The reaction solution was dropped into 2 L of anhydrousacetone (from Wako Pure Chemical Industries, Ltd.), and a precipitatewas recovered by filtration through a suction funnel. The precipitatewas washed with 1 L of anhydrous acetone, followed by vacuum drying.Thus the active esterified pectin was obtained. Incidentally, the ratiosof Z/X and Y/X were as follows:Z/X=1.0 and Y/X=1.0

Example 2 Preparation of Active Esterified Hyaluronic Acid

In 200 g of DMS was added 2.0 g of acid-type hyaluronic acid (containing2.15 mmol/g of carboxy group) prepared in (I)-(2), and dissolved bystirring at 25° C. for 15 hours. The resulting solution was added 0.575g (5.0 mmol) of NHS and 0.958 g (5.0 mmol) of EDC, followed by stirringat 25° C. for 24 hours. The reaction solution was dropped into 2 L ofdiethyl ether (from Wako Pure Chemical Industries, Ltd.), andprecipitate was recovered by filtration through a suction funnel. Theprecipitate was washed with 1 L of tetrahydrofuran (from Wako PureChemical Industries, Ltd.), followed by vacuum drying. Thus the activeesterified hyaluronic acid was obtained. Incidentally, the ratios of Z/Xand Y/X were as follows:Z/X=1.0 and Y/X=1.0

Example 3 Preparation of Active Esterified CM Dextran A1

In 200 g of DMS was added 2.0 g of acid-type dextran A (containing 1.01mmol/g of carboxymethyl group) prepared in (I)-(3), and dissolved bystirring at 100° C. for 15 hours. The resulting solution was added 2.325g (20.2 mmol) of NHS and 1.162 g (6.06 mmol) of EDC, followed bystirring at 25° C. for 24 hours. The reaction solution was dropped into2 L of anhydrous acetone, and precipitate was recovered by filtrationthrough a suction funnel. The precipitate was washed with 1 L ofanhydrous acetone, followed by vacuum drying. Thus the active esterifiedCM dextran A1 was obtained. Incidentally, the ratios of Z/X and Y/X wereas follows:Z/X=3.0 and Y/X=10

Example 4 Preparation of Active Esterified CM Dextran A2

The same procedure as in (I)-(3) was repeated except that 1,162 g (6.06mmol) of EDC was changed to 1.936 g (10.1 mmol) of EDC to give theactive esterified CM dextran A2. Incidentally, the ratios of Z/X and Y/Xwere as follows:Z/X=5.0 and Y/X=10

Example 5 Preparation of Active Esterified CM Dextran A3

The same procedure as in (I)-(3) was repeated except that 1.162 g (6.06mmol) of EDC was changed to 2.325 g (20.2 mmol) of EDC to give theactive esterified CM dextran A2. Incidentally, the ratios of Z/X and Y/Xwere as follows:Z/X=10 and Y/X=10

Example 6 Preparation of Active Esterified CM Dextran B

In 200 g of DMS was added 2.0 g of acid-type dextran B (containing 0.29mmol/g of carboxymethyl group) prepared in (I)-(4), and dissolved bystirring at 100° C. for 15 hours. The resulting solution was added 1.12g (5.8 mmol) of NHS and 0.333 g (2.06 mmol) of EDC, followed by stirringat 25° C. for 24 hours. The reaction solution was dropped into 2 L ofanhydrous acetone, and precipitates were recovered by filtration througha suction funnel. The precipitate was washed with 1 L of anhydrousacetone, followed by vacuum drying. Thus the active esterified CMdextran B was obtained. Incidentally, the ratios of Z/X and Y/X were asfollows:Z/X=5.0 and Y/X=10

Example 7 Preparation of Active Esterified CM Pullulan

In 200 g of DMS was added 2.0 g of acid-type CM pullulan (containing1.28 mmol/g of carboxymethyl group) prepared in (I)-(5), and dissolvedby stirring at 100° C. for 15 hours. The resulting solution was added0.294 g (2.56 mmol) of NHS and 0.491 g (2.56 mmol) of EDC, followed bystirring at 25° C. for 24 hours. The reaction solution was dropped into2 L of anhydrous acetone, and precipitate was recovered by filtrationthrough a suction funnel. The precipitate was washed with 1 L ofanhydrous acetone, followed by vacuum drying. Thus the active esterifiedCM pullulan obtained. Incidentally, the ratios of Z/X and Y/X were asfollows:Z/X=0.5 and Y/X=1.0

(IV) Calculation of the NHS-introducing Ratio of Active EsterifiedPolysaccharide (Polysaccharide Derivative)

The NHS-introducing ratio was calculated for the active esterifiedpolysaccharide (polysaccharide derivative) prepared in Examples 1 to 7in (III). The NHS-introducing ratio is defined as the ratio of theamount of active ester group in the resulting polysaccharide derivativeto the amount of carboxy group or carboxymethyl group in a unit byweight of the starting polysaccharide to be the raw material for thepolysaccharide derivative.

In order to make a calibration curve for N-hydroxysuccinimide (NHS),0.1, 0.2, 0.5, 1.0, 2.5, 5.0, and 10 mM of NHS standard aqueoussolutions were prepared. To 1 mL of each standard aqueous solution ofNHS was added 0.2 mL of 2N aqueous solution of sodium hydroxide,followed by stirring at 60° C. with heating for 10 minutes. Afterstanding to cool, 1.5 mL of 0.85 N hydrochloric acid and 0.75 mL of0.05% FeCl₃/1 N hydrochloric acid solution were added, followed bymeasuring for absorbance at a wavelength of 500 nm by using aspectrophotometer (FeCl₃ from Wako Pure Chemical Industries, Ltd.). Theconcentration of each aqueous solution of NHS on the X axis, and theabsorbance on the Y axis were plotted and then linearised, andconsequently the equation (2) to calculate the concentration of NHS wasobtained as follows.Y=αX+β  (2)where,

X: concentration of NHS (mM)

Y: absorbance at a wavelength of 500 nm

-   -   α=0.102 (gladient)    -   β=0.0138 (intercept)    -   r=0.991 (correlation coefficient)

The content (C mmol) of NHS group in the sample below mentioned can beobtained by multiplying the value of X (mM) calculated from theabsorbance by the volume (3.45 mL) of the solution used for measurement.

Next, 0.01 g of the active esterified polysaccharide prepared inExamples 1 to 7 was weighed and added in 1 mL of pure water, followed bystirring at 25° C. for 3 hours, and then 0.2 mL of 2 N aqueous solutionof sodium hydroxide was added thereto, followed by stirring at 60° C.for 10 minutes. After cooling to the room temperature, 1.5 mL of 0.85 Nhydrochloric acid was added. The resulting solution which includesinsolubles was filtered through filter cotton to remove insolubles, andwas measured for absorbance at a wavelength of 500 nm, after 0.75 mL of0.05% FeCl₃ solution in 1 N hydrochloric acid was added thereto. If themeasured absorbance was higher than that obtained when the concentrationof the NHS standard solution was 5 mM, the solution was diluted withpure water. (The dilution ratio H.) The content (C mmol) of NHS group inthe active esterified polysaccharide was calculated from the measuredabsorbance by using the equation (2) to calculate the concentration ofNHS. And then, the NHS-introducing ratio of the active esterifiedpolysaccharide was determined by using the equation (3) below. Theresults are shown in Table 2.NHS-introducing ratio (%)={(C×H)/0.01}/B×100  (3)where,

B: The total amount of carboxy group in the starting polysaccharide foran active esterified polysaccharide (mmol/g)

C: The amount of NHS group in an active esterified polysaccharide (mmol)

TABLE 2 NHS- Self- Acid-type intro- cross- Bond starting ducing link-strength poly- ratio ablity (g/ saccharide Z/X Y/X (%) (+/−) cm²) (Pa)Example 1 Pectin 1 1 25.7 + 70 6.9 Example 2 Hyaluronic 1 1 8.4 + 50 4.9acid Example 3 CM dextran 3 10 27.7 + 85 8.3 A Example 4 CM dextran 5 1029.7 + 90 8.3 A Example 5 CM dextran 10 10 32.7 + 100 9.8 A Example 6 CMdextran 5 10 15.3 + 120 12 B Example 7 CM pullulan 0.5 1 7.8 + 80 7.8

(V) Self-crosslinkability of Active Esterified Polysaccharide Derivative

The active esterified polysaccharides obtained in Examples 1 to 7 in(III) were tested for self-crosslinkability. 0.2 g of active esterifiedpolysaccharide was weighed in a 10-mL volume of clean test tube (“RaruboLT-15100” from Terumo), and 1 mL of pure water was added thereto andmixed. Then, 1 mL of 8.3% (w/v) aqueous solution of sodium hydrogencarbonate (sodium hydrogen carbonate from Wako Pure Chemical Industries,Ltd.) as a pH adjusting agent was added and mixed at about 2,000 rpm forabout 1 minute by using a test tube mixer (MT-31 from Yamato ScientificCo., Ltd.). The state of the content of the test tube was visuallyobserved before and after mixing. One which has fluidity after mixingsame as the content of the test tube before mixing was regarded as“without self-crosslinkablility (−)”, and one in which the content ofthe test tube formed an agglomerated matrix (hydrogel) was regarded as“with self-crosslinkability (+)”. The results are shown in Table 2above.

(VI) Adhesion Test of Active Esterified Polysaccharide Derivative

An adhesion test in vitro with a fresh integument (pig skin) taken froma Yorkshire edible pig was carried out to determine the adhesionperformance of the active esterified polysaccharide derivatives obtainedin Examples 1 to 7.

A strip specimen with 1 cm in width by 5 cm in length was cut out fromthe pig skin. The dermis of the pig skin was exposed, and the exposedsurface was used as the adherend. The area of adhesion was defined with1 cm by 1 cm. To the adherend was applied 100 μL of solution prepared bydissolving 0.2 weighed of the active esterified polysaccharide obtainedin Examples 1 to 7 in 1 mL of pure water, and was further applied 10 μLof 8.3% (w/v) aqueous solution of sodium hydrogen carbonate, followed bymixing them. On the adherend, another pig skin strip was placed andloaded with 50 gf/cm² (ca. 4.9 Pa) for 1 minute. After standing for 5minutes, the adhesion test was carried out by pulling two-adhered sheetsof pig skin strip in the longitudinal direction and in oppositedirection with one another at a cross-head speed of 100 mm/min by usinga autograph (tensile tester). The tensile strength required to peel offthe two pig skin strips was regarded as the adhesive strength. Theresults are shown in Table 2.

The same experiments as in Examples 1 to 7 were repeated except that8.3% (w/v) aqueous solution of sodium hydrogen carbonate (pH 8.3)(sodium hydrogen carbonate from Wako Pure Chemical Industries, Ltd.) wasreplaced by 1 mol/L aqueous solution of disodium phosphate (pH 9.1)(disodium phosphate from Wako Pure Chemical Industries, Ltd.). Inresult, all of that obtained in examples 1 to 7 possessedself-crosslinkability (+).

The same experiments as in Examples 5 and 7 were repeated except that 10μL of 8.3% (w/v) aqueous solution of sodium hydrogen carbonate wasreplaced by 10 μL of 1 mol/L aqueous solution of disodium phosphate (pH9.1). In result, the adhesive strength of ones that obtained in examples5 and 7 were 115 g/cm²=11.3 Pa and 95 g/cm²=9.4 Pa, respectively.

(VII) Preparation of Polysaccharide Composition

To an active esterified polysaccharide derivative was added a polymer(C) to prepare a polysaccharide composition.

Example 8 Active Esterified Pectin Composition 1

0.2 g-weighed of the active esterified pectin prepared in Example 1 wasadded in 1 mL of pure water, and mixed to prepare a main solution.Meanwhile, In 1 mL of 8.3% (w/v) aqueous solution of sodium hydrogencarbonate at (pH 8.3) was added 0.2 g weighed of pentaerythritol-typepolyethylene glycol (PEG) derivative having a thiol group on fourterminals (Sunbrite PTE-10TSH from NOF Corporation, having aweight-average molecular weight of 10,000), and mixed to prepare aassociate solution. 1 mL of the main solution and 1 mL of the associatesolution were mixed in a test tube to prepare the polysaccharidecomposition. The polysaccharide composition has fluidity just aftermixing of the main solution and the associate solution, however, thecontent of the test tube formed an agglomerated matrix (hydrogel) aftermixing for about 1 minute at about 2,000 rpm by using a test tube mixer.

Example 9 Active Esterified Pectin Composition 2

The active esterified pectin in example 1 was used. The same procedureas in Example 8 was repeated to prepare the polysaccharide compositionexcept that the pentaerythritol-type polyethylene glycol derivative usedin example 8 was replaced by glycerol-type polyethylene glycolderivative having an amino group on eight terminals (Sunbrite HGEO-20TEAfrom NOF Corporation, having a weight-average molecular weight of10,000).

Example 10 Active Esterified Pectin Composition 3

The active esterified pectin in example 1 was used. The same procedureas in Example 8 was repeated to prepare the polysaccharide compositionexcept that the pentaerythritol-type polyethylene glycol derivative usedin example 8 was replaced by bovine serum albumin (from Sigma).

Example 11 Active Esterified Pectin Composition 4

The active esterified pectin in example 1 was used. The same procedureas in Example 8 was repeated to prepare the polysaccharide compositionexcept that the pentaerythritol-type polyethylene glycol derivative usedin example 8 was replaced by 0.02 g of pectin (GENU Pectin USP-H from CPKelco).

Example 12 Active Esterified Dextran A1 Composition 1

In 1 mL of pure water was added 0.2 g-weighed of the active esterifieddextran A1 prepared in Example 3, and mixed to prepare a main solution.Meanwhile, in 1 mL of 8.3% (w/v) aqueous solution of sodium hydrogencarbonate (pH 8.3) was added 0.2 g-weighed of pentaerythritol-typepolyethylene glycol (PEG) derivative having a thiol group on fourterminals (with a weight-average molecular weight of 20,000) (see, Poly(ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications,compiled by J. Milton Harris, Plenum Press, NY (1992)), and mixed toprepare a associate solution. 1 mL of the main solution and 1 mL of theassociate solution were mixed in a test tube to prepare thepolysaccharide composition. The polysaccharide composition has fluidityjust after mixing of the main solution and the associate solution,however, the content of the test tube formed an agglomerated matrix(hydrogel) after mixing for about 1 minute at about 2,000 rpm by using atest tube mixer.

Example 13 Active Esterified Dextran A1 Composition 2

In 1 mL of pure water was added 0.2 g-weighed of the active esterifieddextran A1 prepared in Example 3, and mixed to prepare a main solution.Meanwhile, in 1 mL of 8.3% (w/v) aqueous solution of sodium hydrogencarbonate (pH 8.3) was added 0.2 g-weighed of pentaerythritol-typepolyethylene glycol (PEG) derivative having an amino group on fourterminals (with a weight-average molecular weight of 10,000) (see, Poly(ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications,compiled by J. Milton Harris, Plenum Press, NY (1992)), and mixed toprepare a associate solution. 1 mL of the main solution and 1 mL of theassociate solution were mixed in a test tube to prepare thepolysaccharide composition. The polysaccharide composition has fluidityjust after mixing of the main solution and the associate solution,however, the content of the test tube formed an agglomerated matrix(hydrogel) after mixing for about 1 minute at about 2,000 rpm by using atest tube mixer.

Example 14 Active Esterified Dextran A1 Composition 3

In 1 mL of pure water was added 0.2 g-weighed of the active esterifieddextran Al prepared in Example 3, and mixed to prepare a main solution.Meanwhile, in 1 mL of 8.3% (w/v) aqueous solution of sodium hydrogencarbonate (pH 8.3) was added 0.2 g-weighed of pentaerythritol-typepolyethylene glycol (PEG) derivative having an amino group on fourterminals (with a weight-average molecular weight of 20,000) (see, Poly(ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications,compiled by J. Milton Harris, Plenum Press, NY (1992)), and mixed toprepare a associate solution. 1 mL of the main solution and 1 mL of theassociate solution were mixed in a test tube to prepare thepolysaccharide composition. The polysaccharide composition has fluidityjust after mixing of the main solution and the associate solution,however, the content of the test tube formed an agglomerated matrix(hydrogel) after mixing for about 1 minute at about 2,000 rpm by using atest tube mixer.

Example 15 Active Esterified Pullulan Composition

In 1 mL of pure water was added 0.2 g-weighed of the active esterifiedpullulan prepared in Example 7, and mixed to prepare a main solution.Meanwhile, in 1 mL of 8.3% (w/v) aqueous solution of sodium hydrogencarbonate (pH 8.3) was added 0.2 g-weighed of pentaerythritol-typepolyethylene glycol (PEG) derivative having an amino group on fourterminals (with a weight-average molecular weight of 20,000) (see, Poly(ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications,compiled by J. Milton Harris, Plenum Press, NY (1992)), and mixed toprepare a associate solution. 1 mL of the main solution and 1 mL of theassociate solution were mixed in a test tube to prepare thepolysaccharide composition. The polysaccharide composition has fluidityjust after mixing of the main solution and the associate solution,however, the content of the test tube formed an agglomerated matrix(hydrogel) after mixing for about 1 minute at about 2,000 rpm by using atest tube mixer.

(VIII) Adhesion Test (1) of Active Esterified Polysaccharide Composition

In order to determine the adhesivility of the polysaccharidecompositions prepared in Examples 8 to 11, an adhesion test was carriedout in vitro in the same way as in (VI). For the active esterifiedpolysaccharide compositions obtained in Examples 8 to 11, the adhesiontest was carried out by applying 50 μL of the main solution and 50 μL ofthe associate solution on the adherend, and mixing them. The results areshown in Table 3 below.

TABLE 3 Active esterified polysaccharide Bond strength derivative g/cm²Pa From Example 1 Example 8 82 8.0 Example 9 From Example 1Glycerol-type PEG 105 10 derivative Example 10 From Example 1 Bovineserum albumin 120 12 Example 11 From Example 1 Pectin 98 9.6

(VIII) Adhesion Test (2) of Active Esterified Polysaccharide Composition

In order to determine the adhesivility of the polysaccharidecompositions prepared in Examples 12 to 15, an adhesion test was carriedout in vitro in the same way as in (VI). For the active esterifiedpolysaccharide compositions obtained in Examples 12 to 15, the adhesiontest was carried out by applying 50 μL of the main solution and 50 μL ofthe associate solution on the adherend, and mixing them. The results areshown in Table 4 below.

TABLE 4 Active esterified adhesive polysac- strength charide (g/derivative Polymer added cm²) (Pa) Example 12 Example 3Pentaerythritol-type PEG 103 10.1 derivative having a thiol group onfour terminals (with a weight average molecular weight of 20,000)Example 13 Example 4 Pentaerythritol-type PEG 86 8.4 derivative havingan amino group on four terminals (with a weight average molecular weightof 10,000) Example 14 Example 5 Pentaerythritol-type PEG 98 9.6derivative having an amino group on four terminals (with a weightaverage molecular weight of 20,000) Example 15 Example 7Pentaerythritol-type PEG 128 12.6 derivative having an amino group onfour terminals (with a weight average molecular weight of 20,000)

(IX) Preparation of Medical Treatment Material Example 16 (1) LiquidMedical Treatment Material 1 Containing Active Esterified Pectin

In 1 mL of pure water was added 0.2 g of active esterified pectinobtained in Example 1, and mixed to form a aqueous solution as a mainsolution. An 8.3% (w/v) aqueous solution of sodium hydrogen carbonatewas used as the pH adjusting solution. A liquid medical treatmentmaterial 1 which composed of the main solution and the pH adjustingsolution was prepared. The main solution and the pH adjusting solutionmay be used by mixing them at the applying site.

Example 17 (2) Liquid Medical Treatment Material 2 Containing ActiveEsterified Pectin

In 1 mL of pure water was added 0.2 .g-weighed of active esterifiedpectin obtained in Example 1, and mixed to form a aqueous solution as amain solution. Meanwhile, in 1 mL of 8.3% (w/v) aqueous solution ofsodium hydrogen carbonate was added 0.2 g-weighed of thepentaerythritol-type polyethylene glycol derivative having a thiol groupon four terminals, and mixed to form a aqueous solution as a associatesolution. A liquid medical treatment material 2 which composed of themain solution and the associate solution was prepared. The main solutionand the associate solution may be used by mixing them at the applyingsite.

Example 18 (3) Powdery Medical Treatment Material Containing ActiveEsterified CM Dextran A1

A powdery medical treatment material was prepared by mixing from 0.5 gof powder obtained by freeze-crushing the active esterified CM dextranAobtained in Example 3 and 0.05 g of sodium hydrogen carbonate in powderform. This medical treatment material can be applied to the applicationsite by dusting and mixing them.

Example 19 (4) Powdery Medical Treatment Material Kit Containing ActiveEsterified CM Dextran A1

A powdery medical treatment material kit was prepared , which wascomposed of 0.5 g of powder obtained by freeze-crushing the activeesterified CM dextran A1 obtained in Example 3 and 100 μL of 8.3% (w/v)aqueous solution of sodium hydrogen carbonate otherwise prepared. Thismedical treatment material kit can be applied to the application site bydusting and mixing them

Example 20 (5) Sheet-like Medical Treatment Material and Kit 1Containing Active Esterified Pectin

30 mL of 5% (w/v) aqueous solution of the active esterified pectinobtained in Example 1 was prepared and spread on a plastic dish with 5cm×5 cm, followed by being frozen at −50° C. in a deep freezer and thendried in remaining its frozen state under reduced pressure using avacuum dryer. Thus there was obtained a sheet-like medical treatmentmaterial with about 5 mm thick. In addition, 1 mL of 8.3% (w/v) aqueoussolution of sodium hydrogen carbonate otherwise prepared was composedwith the sheet-like medical treatment material to set up a sheet-likemedical treatment material kit (1). The sheet-like medical treatmentmaterial may be put on the application site and the 8.3% (w/v) aqueoussolution of sodium hydrogen carbonate is applied thereon.

Example 21 (6) Sheet-like Medical Treatment Material and Kit 2Containing Active Esterified Pectin

The sheet-like medical treatment material prepared in Example 20 wasused. A coating solution was prepared by dissolving 5 g of glycerol-typepolyethylene glycol derivative having an amino group on eight terminalsin 100 mL of chloroform (from Wako Puke Chemical Industries, Ltd.). Thesheet-like medical treatment material prepared in Example 20 wasimmersed in the coating solution for 1 minute, followed by air drying at25° C. for 3 hours. Thus there was obtained a sheet-like medicaltreatment material comprising the active esterified pectin and thepolysaccharide composition of glycerol-type polyethylene glycolderivative having an amino group on eight terminals. In addition, Asheet-like medical treatment material kit 2 was also prepared which wascomposed of 1 mL of 8.3% (w/v) aqueous solution of sodium hydrogencarbonate otherwise prepared and the above-mentioned sheet-like medicaltreatment material. The sheet-like medical treatment material may be puton the application site and the 8.3% (w/v) aqueous solution of sodiumhydrogen carbonate is applied thereon.(X) Evaluation test for hemostaticperformance by using small animals

A rat (Jcl: SD strain, from Clea Japan, Inc.) was subjected to generalanesthesia with Nembutal (from Dainabott) administered by intramuscularinjection and had its abdomen cut open so that its spleen was exposed.The surface of the spleen was made an incision about 2 mm long with ascalpel so as to make a bleeding model. Evaluation tests of hemostaticperformance for medical treatment materials prepared in Examples 16 to21 were carried out to observe hemorrhagic states three minutes aftertheir application, and resulted in performances of hemostasis in all themedical treatment materials. These medical treatment materials wereprepared in a simple manner just before their application, and did notrequire any special apparatus.

It is designated in Table 2 that the polysaccharide derivativesaccording to the present invention (Examples 1 to 7) have an adhesivestrength ranging form 50 to 120 (g/cm²) within the range 30 to 150(g/cm²) which is required generally for a hemostatic material and amedical adhesive, and also within the range 50 to 120 (g/cm²) which issuitable for such uses. Likewise, the polysaccharide compositionsaccording to the present invention (Examples 8 to 15) also have anadhesive ranging form 82 to 128 (g/cm²), and are suitable for use as ahemostatic material and a medical adhesive.

The present invention is described in detail by way of examples, below.However, these examples are demonstrated by way an example, thus thepresent invention is not limited thereto.

INDUSTRIAL APPLICABILITY

The polysaccharide derivative and composition of the present invention,in using as an adhesive for tissues, is a sufficient bond strength thatmeets clinical requirements. They avoid the risk of infection becausethey have the backbone of natural or artificial polysaccharide. Theircomponents in themselves or their decomposition products have a lowlevel of toxicity. The polysaccharide derivative of the presentinvention does not need complex steps for preparation at the time ofuse. It can be prepared readily and simply without requiring specialapparatus at the time of use. The polysaccharide derivative may beprovided alone as the polysaccharide composition; therefore, it willfind a large variety of uses. Since the polysaccharide composition ofthe present invention employs the polysaccharide derivative of thepresent invention, it does not impair the characteristic properties ofthe present invention.

The polysaccharide derivative and composition of the present inventioncan be fabricated into powder, sheet, granules, or any other forms.Therefore, they can be used in various ways according to the object oftheir use. The polysaccharide derivative and composition of the presentinvention can be produced simply by mixing with heating necessaryreagents, without requiring special apparatus. Owing to theabove-mentioned characteristic properties, the polysaccharide derivativeand composition of the present invention are suitable for use as themedical treatment material such as hemostatic agent and adhesive.

1. A crosslinkable polysaceharide composition comprising anuncrosslinked polysaceharide derivative (A) and a pH adjusting agent(B), wherein said polysaceharide derivative (A) has at least one activeester group containing a N-hydroxylamine-based electrophilic moietywhich is introduced to a carboxy group at a side chain of theacid-containing polysaceharide, and may form a covalent bond through areaction with an active hydrogen-containing group selected from thegroup consisting of a hydroxy group, an amino group, and a thiol groupunder alkaline conditions in the presence of moisture, and which is apowder mixture comprising the polysaceharide derivative (A) and the pHadjusting agent (B).
 2. The composition according to claim 1, whichfurther comprises a polymer other than said polysaceharide derivative(A).
 3. A medical treatment material which comprises the compositionaccording to claim
 1. 4. The medical treatment material according toclaim 3, which is a hemostatic agent and/or biomedical adhesive.